Bioadhesive for corneal repair

ABSTRACT

The present invention provides compositions and methods for repair and reconstruction of defects and injuries to the cornea.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase Entry of International PatentApplication No. PCT/US2017/016917 filed on Feb. 8, 2017 which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.62/292,752 filed Feb. 8, 2016, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE DISCLOSURE

The field of the disclosure relates to improved tissue adhesives for usein repairing corneal injuries and defects. These tissue adhesivescomprise elastic biopolymers which are biocompatible, biodegradable,transparent, strongly adhesive to corneal tissue, and have a smoothsurface and biomechanical properties similar to the cornea.

BACKGROUND

Ocular trauma is common, accounting for nearly 5% of blindness in thegeneral population.¹ The current standards of care for repair of cornealinjuries, including lacerations, structural defects, and thinning, oftenrequire suturing, tissue/patch grafting, and/or glue application.However, these standard procedures are associated with substantialdrawbacks, including: (1) corneal sutures are foreign bodies that canserve as risk factors for microbial entrapment and infection,inflammation, and neovascularization; (2) corneal sutures often induceregular or irregular astigmatism, leading to impaired visual acuity; (3)corneal transplantation and patch grafting require donor tissues, whichmay not be readily available; and (4) use of allogeneic tissues forgrafting carries a high risk for immune reactions in acutely injuredinflamed eyes. Thus, even if the structural repair is adequate with thecurrent standards of care, the visual outcomes are often notsatisfactory.^(2,3) In addition, (5) application of any of the currentlyavailable glue/adhesive technologies for temporizing injured eyes untilmore definitive care can be offered has its own inherent limitations.

To circumvent some of the limitations of the current surgical approachesin corneal repair, the use of adhesives may be considered for fastrepair of corneal injuries. However, currently there is no approvedadhesive for filling corneal defects. The only approved sealant in theU.S., ReSure®, is for sealing corneal incisions of cataract surgery, andhas not been designed for filling corneal defects and falls off quickly(usually in less than 3 days).⁴ OcuSeal®, a sealant used in Europe, isalso utilized for protecting corneal incisions but not filling cornealdefects and also detaches quickly. For this reason, cyanoacrylate glue,which is approved for repair of skin wounds, is currently used as“off-label” for treating many ophthalmic settings such as cornealperforations, impending perforations and progressive corneal thinningdisorders.^(5,6) However, cyanoacrylate glue has several majordrawbacks, including:

(1) Low biocompatibility, with cytotoxic effects on the cornea and otherocular tissues (risk of cataract formation and retinopathy if it entersthe eye);⁷⁻¹¹ (2) lack of transparency, precluding good vision andimpairing view of retrocorneal structures; (3) risk of secondaryinfection due to high porosity;¹² (4) difficult to control itsapplication, with glue potentially falling off unpredictably; (5) roughsurface requiring contact lens wear, which adds additional infectionrisk; and (6) it does not integrate with corneal tissue.

Because existing adhesives for corneal repair have major drawbacks,there is an unmet need for an adhesive for the repair and regenerationof corneal injuries that can meet the following requirements: (1) easyapplication; (2) biocompatible without causing any toxicity,inflammation, or neovascularization; (3) transparent so as to enablerestoration of vision as quickly as possible; (4) ability to rapidlyseal the corneal wound; (5) permitting corneal cells to integrate withthe bioadhesive to facilitate tissue regeneration (6) biomechanicalproperties (rigidity and elasticity) similar to the cornea; (7) strongadhesion to corneal tissue including good stability and high retention;and (8) smooth surface to reduce the need for bandage contact lens andminimize surface area for microbial adhesion.

Photopolymerization of methacryloyl-substituted gelatin is aninexpensive and technically simple approach to fabricate hydrogels forbiomedical applications.^(14, 38-40) The cytocompatibility ofmethacryloyl-substituted gelatin has been previously proven, suggestingit has potential to be implanted into a living organism.⁴¹⁻⁴² However,its actual function as a corneal repair material has not been evaluatedyet. Moreover, the mechanical properties of methacryloyl-substitutedgelatin have not been thoroughly investigated, so it is unknown if it issuitable to serve as a bioadhesive for corneal repair.

SUMMARY

Certain aspects of the present invention are directed to compositionsfor corneal reconstruction comprising a methacryloyl gelatin (GelMA)prepolymer, a visible light activated photoinitiator, and apharmaceutically acceptable carrier. In some embodiments, themethacryloyl-substituted gelatin has a degree of methacryloylsubstitution between 30% and 85%, between 60% and 85%, or between 70%and 80%. Methacryloyl gelatin is also referred to as methacryloylsubstituted gelatin herein.

In some embodiments, the methacryloyl-substituted gelatin comprisesmethacrylamide substitution and methacrylate substitution, and the ratioof methacrylamide substitution to methacrylate substitution is between80:20 and 99:1, between 90:10 and 98:2, or between 93:7 and 97:3.

In some embodiments, the methacryloyl-substituted gelatin is present ata concentration between 5% and 25% (w/v), between 17% and 55% (w/v),between 17% and 23% (w/v), between 5% and 15% (w/v), between 8% and 12%(w/v), of about 20% (w/v) or of about 10% (w/v).

In some embodiments, the visible light activated photoinitiator isselected from the group consisting of: Eosin Y, triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ),1-phenyl-1,2-propadione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphineoxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide(Ir819), 4,4′-bis(dimethylamino)benzophenone,4,4′-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one,4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene,diphenyl(2,4,6 trimethylbenzoyl)phosphineoxide/2-hydroxy-2-methylpropiophenone (50/50 blend), dibenzosuberenone,(benzene) tricarbonylchromium, resazurin, resorufin,benzoyltrimethylgermane (Ivocerin®), derivatives thereof, and anycombination thereof.

Preferably, the visible light activated photoinitiator comprises amixture of Eosin Y, triethanolamine, and vinyl caprolactam. In someembodiments of the photoinitiator mixture, the concentration of Eosin Yis between 0.0125 and 0.5 mM, and/or the concentration oftriethanolamine is between 0.1 and 2% w/v, and/or the concentration ofvinyl caprolactam is between 0.05 and 1.5% w/v.

In some embodiments of the photoinitiator mixture, the concentration ofEosin Y is between 0.025 and 0.15 mM, and/or the concentration oftriethanolamine is between 0.2 and 1.6% w/v, and/or and theconcentration of vinyl caprolactam is between 0.09 and 0.8% w/v. In someembodiments of the photoinitiator mixture, the concentration of Eosin Yis between 0.025 and 0.15 mM, and/or the concentration oftriethanolamine is between 0.2 and 1.6% w/v, and/or the concentration ofvinyl caprolactam is between 0.09 and 0.8% w/v. In some embodiments ofthe photoinitiator mixture, the concentration of Eosin Y is between 0.05and 0.08 mM, and/or the concentration of triethanolamine is between 0.4and 0.8% w/v, and/or the concentration of vinyl caprolactam is between0.18 and 0.4% w/v. In some embodiments of the photoinitiator mixture,the concentration of Eosin Y is about 0.05 mM, and/or the concentrationof triethanolamine is about 0.4% w/v, and/or the concentration of vinylcaprolactam is about 0.4% w/v. In some embodiments of the photoinitiatormixture, the concentration of Eosin Y is between 0.5 and 0.5 mM, and/orthe concentration of triethanolamine is between 0.5 and 2% w/v, and/orthe concentration of vinyl caprolactam is between 0.5 and 1.5% w/v. Insome embodiments of the photoinitiator mixture, the concentration ofEosin Y is about 0.1 mM, the concentration of triethanolamine is about0.5% w/v, and the concentration of vinyl caprolactam is about 0.5% w/v.

In some embodiments, the composition further comprises corneal cells.Exemplary, corneal cells include, but are not limited to, epithelialcells, endothelial cells, keratocytes, and any combinations thereof.

In some embodiments, the composition further comprises a therapeuticagent. Exemplary therapeutic agents for inclusion in the compositionsinclude, but are not limited to, an antibacterial, an anti-fungal, ananti-viral, an anti-acanthamoebal, an anti-inflammatory, animmunosuppressive, an anti-glaucoma, an anti-VEGF, a growth factor, orany combination thereof.

Certain aspects of the present invention are directed to compositionsfor corneal reconstruction comprising a crosslinkedmethacryloyl-substituted gelatin hydrogel and a pharmaceuticallyacceptable carrier, wherein the crosslinked methacryloyl-substitutedgelatin hydrogel has a degree of methacryloyl substitution between 30%and 85% and a concentration between 5% and 25% (w/v) in thepharmaceutically acceptable carrier. These compositions are alsoreferred to as cross-linked compositions herein. Further, suchcompositions are also referred to as Gel-CORE herein.

In some embodiments, the crosslinked methacryloyl-substituted gelatinhydrogel has a degree of methacryloyl substitution between 60% and 85%and a concentration between 8% and 12% (w/v), or a degree ofmethacryloyl substitution between 70% and 80% and a concentration ofabout 10% (w/v). In some embodiments, the crosslinkedmethacryloyl-substituted gelatin hydrogel has a degree of methacryloylsubstitution between 60% and 85% and a concentration between 17% and 25%(w/v), or a degree of methacryloyl substitution between 70% and 80% anda concentration of about 20% (w/v).

In some embodiments, the cross-linked composition has a Young's Modulusof 190-260 kPa. In some embodiments, the cross-linked composition has aYoung's Modulus of 110-140 kPa.

In some embodiments, the cross-linked composition has an elastic modulusof 5-50 kPa.

In some embodiments, the cross-linked composition has a compressivemodulus of 5-320 kPa. In some embodiments, the composition has acompressive modulus of 5-160 kPa. In still some other embodiments, thecomposition has a compressive modulus of 125-175 kPa.

In some embodiments, the cross-linked composition has wound closurestrength of ≥40 kPa.

In some embodiments, the cross-linked composition has a shear resistancestrength of ≥10 kPa. In some embodiments, the cross-linked compositionhas a shear resistance strength of ≥100 kPa.

In some embodiments, the cross-linked composition has a burst pressureof ≥15 kPa.

In some embodiments, the cross-linked composition further comprises atherapeutic agent. Some exemplary therapeutic agents are anantibacterial, an anti-fungal, an anti-viral, an anti-acanthamoebal, ananti-inflammatory, an immunosuppressive, an anti-glaucoma, an anti-VEGF,a growth factor, or any combination thereof.

In some embodiments, the cross-linked composition further comprisescorneal cells. Preferred corneal cells include endothelial cells,keratocytes, or a combination thereof.

In some embodiments, the cross-linked composition is substantiallytransparent.

In some embodiments, the cross-linked composition has a substantiallysmooth surface.

Certain aspects of the present invention are directed to methods forcorneal reconstruction, comprising the steps of: applying a compositioncomprising a methacryloyl-substituted gelatin, a visible light activatedphotoinitiator, and a pharmaceutically acceptable carrier to a cornealdefect; and exposing the composition to visible light. In someembodiments of the method, the methacryloyl-substituted gelatin has adegree of methacryloyl substitution between 30% and 85%, between 60% and85%, or between 70% and 80%.

In some embodiments of the method, the methacryloyl-substituted gelatincomprises methacrylamide substitution and methacrylate substitution, andthe ratio of methacrylamide substitution to methacrylate substitution isbetween 80:20 and 99:1, between 90:10 and 98:2, or between 92:8 and97:3.

In some embodiments of the method, the methacryloyl-substituted gelatinis present at a concentration between 5% and 25% (w/v), between 17% and55% (w/v), between 17% and 23% (w/v), between 5% and 15% (w/v), between8% and 12% (w/v), of about 20% (w/v) or of about 10% (w/v).

In some embodiments of the method, the visible light activatedphotoinitiator is selected from the group consisting of: Eosin Y,triethanolamine, vinyl caprolactam,dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propadione(PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO),bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819),4,4′-bis(dimethylamino)benzophenone, 4,4′-bis(diethylamino)benzophenone,2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone,phenanthrenequinone, ferrocene, diphenyl(2,4,6trimethylbenzoyl)phosphine oxide/2-hydroxy-2-methylpropiophenone (50/50blend), dibenzosuberenone, (benzene) tricarbonylchromium, resazurin,resorufin, benzoyltrimethylgermane (Ivocerin®), derivatives thereof, andany combination thereof.

In some embodiments of the method, the visible light activatedphotoinitiator comprises a mixture of Eosin Y, triethanolamine, andvinyl caprolactam. In some embodiments of the photoinitiator mixture,the concentration of Eosin Y is between 0.0125 and 0.5 mM, and/or theconcentration of triethanolamine is between 0.1 and 2% (w/v), and/or theconcentration of vinyl caprolactam is between 0.05 and 1.5% (w/v). Insome embodiments of the method, the concentration of Eosin Y is between0.025 and 0.15 mM, and/or the concentration of triethanolamine isbetween 0.2 and 1.6% (w/v), and/or and the concentration of vinylcaprolactam is between 0.09 and 0.8% w/v. In some embodiments of themethod, the concentration of Eosin Y is between 0.025 and 0.15 mM,and/or the concentration of triethanolamine is between 0.2 and 1.6% w/v,and/or the concentration of vinyl caprolactam is between 0.09 and 0.8%(w/v). In some embodiments of the method, the concentration of Eosin Yis between 0.05 and 0.08 mM, and/or the concentration of triethanolamineis between 0.4 and 0.8% w/v, and/or the concentration of vinylcaprolactam is between 0.18 and 0.4% (w/v). In some embodiments of themethod, the concentration of Eosin Y is about 0.05 mM, and/or theconcentration of triethanolamine is about 0.4% (w/v), and/or theconcentration of vinyl caprolactam is about 0.4% (w/v). In someembodiments of the method, the concentration of Eosin Y is between 0.5and 0.5 mM, and/or the concentration of triethanolamine is between 0.5and 2% (w/v), and/or the concentration of vinyl caprolactam is between0.5 and 1.5% (w/v). In some embodiments of the method, the concentrationof Eosin Y is about 0.1 mM, the concentration of triethanolamine isabout 0.5% (w/v), and the concentration of vinyl caprolactam is about0.5% (w/v).

Generally, a light of any suitable wavelength can be used in the methodof the invention. For example, the composition can be exposed to visiblelight with a wavelength in the range of 450 to 550 nm. Further, exposureto light can be for any desired duration of time. For example, thecomposition can be exposed to visible light for a time period between 10and 300 seconds. In some embodiments, the composition can be exposed tovisible light for a time period between 20 and 120 seconds, or between30 and 60 seconds. In some embodiments, the composition can be exposedto visible light for a time period between 60 seconds and 240 seconds.In some embodiments, the composition can be exposed to visible light fora time period of about 60 seconds, about 120 seconds, about 180 secondsor about 240 seconds. In some preferred embodiments, the composition canbe exposed to visible light for a time period of about 240 seconds.

In some embodiments of the method, the composition further comprises atherapeutic agent, preferably an antibacterial, an anti-fungal, ananti-viral, an anti-acanthamoebal, an anti-inflammatory, animmunosuppressive, an anti-glaucoma, an anti-VEGF, a growth factor, orany combination thereof. In some embodiments, the composition furthercomprises corneal cells, preferably epithelial cells, endothelial cells,keratocytes, or a combination thereof. In some embodiments, thecomposition is substantially transparent. In some embodiments, themethod does not comprise suturing the cornea.

In some embodiments of the method, the composition is a composition forcorneal reconstruction described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an Optical Coherence Tomography (OCT) image of cyanoacrylateglue on a patient's cornea showing its rough surface and high porosity.FIG. 1B is a slit lamp photograph of cyanoacrylate glue showing itsopaqueness. FIGS. 1C-1D are Gel-CORE slit lamp photos and FIG. 1E is anOCT image after ex vivo application to rabbit corneal defect showing itsclarity, and smoothness.

FIG. 2A is a reaction scheme showing fluoraldehyde assay to determineconversion of amine groups in GelMA. The reaction between unmodifiedlysine residues in methacrylic anhydrate (MA)-modified materials and thefluoraldehyde reagents generates blue fluorescent derivatives, whichshow excitation and emission maxima at wavelengths of 340 nm and 455 nm,respectively. FIG. 2B is a bar graph showing conversions of amine groupsin GelMA samples. Gelatin is used as the control.

FIGS. 3A-3E show Fe(III)-hydroxamic acid assay for quantification ofmethacrylate groups in GelMA. FIG. 3A is a schematic diagram showingreactions used for the analysis of methacrylate groups. Step (i) showsthat methacrylate groups in GelMA react with hydroxylamine under basicconditions to generate N-hydroxymethacrylamide in equal molar amount.Step (ii) shows that N-hydroxymethacrylamide forms a colored complexwith Fe(III) ion under acidic conditions. FIG. 3B shows photographs ofan Fe(III) solution before and after the addition of acetohydroxamicacid. The color change indicates the formation of theFe(III)-acetohydroxamic acid complex. FIG. 3C shows normalized UV-Visabsorption spectrum of the Fe(III)-acetohydroxamic acid complex (FeAHA).The absorption peak is centered at 500 nm. FIG. 3D shows a working curvebetween absorbance and FeAHA concentration from a series of standardFeAHA solutions. FIG. 3E shows calculated amounts of methacrylamide andmethacrylate groups in GelMA samples. The ultra-GelMA, high-GelMA,medium-GelMA, and low-GelMA refer to samples prepared with 20% (v/v), 8%(v/v), 5% (v/v), and 0.5% (v/v) MA in the reaction, respectively.

FIGS. 4A-4C show an exemplary Eosin-Y-based photopolymerization systemof the invention. FIG. 4A is a schematic illustration of the radicalgeneration reactions initiated by photo-induced activation of Eosin-Y,which is assisted by TEA. FIG. 4B shows UV-Vis spectra of Eosin-Y at pH7.4 in aqueous solution. FIG. 4C shows visualization of the color changeupon visible light exposure during the hydrogel formation indicatingactivated state of Eosin-Y.

FIGS. 5A-5B show visible light crosslinked GelMA hydrogel with tunable(FIGS. 5A and 5B) mechanical properties—Stress vs strain (FIG. 5A) andTensile Modulus vs TEA concentration (FIG. 5B). (*p<0.05, **p<0.01,***p<0.001 and ****p<0.0001)

FIGS. 6A-6B show visible light crosslinked GelMA hydrogel with tunableswelling ratio varying VC concentrations (FIG. 6A) and TEAconcentrations (FIG. 6B).

FIG. 7A depicts a schematic of the sample preparation for burst pressuretesting. FIG. 7B shows the top view of the burst pressure test setup(porcine intestine is placed between the metal plates). FIG. 7C is a bargraph showing burst pressure data for GelMA hydrogel compared to CoSealand Evicel. GelMA were produced according to Examples 2 and 3.

FIG. 8A depicts a schematic of the modified standard test method forwound closure strength (ASTM F2458-05). FIG. 8B is a bar graph showingadhesive strength of said sealants using the wound closure test atvarying GelMA concentrations and exposure times. GelMA were producedaccording to Examples 2 and 3.

FIGS. 9A-9D show slit lamp photographs (FIGS. 9A and 9C) and OCT images(FIGS. 9B and 9D) immediately after ex vivo application of GelMA torabbit cornea (FIGS. 9A and 9B) and 11 days later (FIGS. 9C and 9D). Ascan be seen, there was excellent retention of GelMA.

FIG. 10A is a photograph showing line scans obtained at different anglesfor OCT imaging. FIG. 10B is an image showing thickness of thebioadhesive which was measured in the center and 1 mm from the center.

FIGS. 11A-11G show in vitro evaluation of GelMA cytocompatiblity andspreading using corneal keratocyte cells. FIGS. 11A-11C showrepresentative live/dead images from keratocytes attached on the surfaceof the Gel-CORE on days 1 (FIG. 11A), 3 (FIG. 11B) and 7 (FIG. 11C).FIGS. 11D-11F show representative images from F-actin/DAPI stainedGEL-CORE containing keratocytes days 1 (FIG. 11D), 3 (FIG. 11E) and 7(FIG. 11F) after encapsulation FIG. 11G shows quantification of cellviability on GEL-CORE over 7 days of culture.

FIGS. 12A-12F are a photographs showing IVCM images of different corneallayers in the rabbit: Superficial epithelium (FIG. 12A), basalepithelium (FIG. 12B), subbasal nerves (FIG. 12C), anterior stromalkeratocytes (FIG. 12D), posterior stromal keratocytes (FIG. 12E), andendothelium (FIG. 12F).

FIGS. 13A-13B are slit lamp photographs and OCT images following GelMAapplication to partial-thickness corneal defects in two rabbit eyes exvivo. A 3 mm partial trephination filled with porcine GelMA crosslinkedwith blue light for 120 seconds.

FIGS. 14A-14C show properties of some exemplary GelMA hydrogels of theinvention. FIG. 14A is a line graph showing representative tensilestrain/stress curves, FIG. 14B is a bar graph showing tensile modulusfor GelMA hydrogels produced at various light exposure times and GelMAconcentrations. FIG. 14C is a bar graph showing strength at break forGelMA hydrogels, crosslinked by using various visible light exposuretimes and GelMA concentrations.

FIGS. 15A-15B show properties of some exemplary GelMA hydrogels of theinvention. FIG. 15A is a line graph showing representative comressivestrain/stress curves.

FIG. 15B is a bar graph showing compression modulus for GelMA hydrogelsproduced at various light exposure times and GelMA concentrations.

FIGS. 16A-16B show the setup (FIG. 16A) and results (FIG. 16B) for exvivo burst pressures of cornea sealed by visible light crosslinked GelMAhydrogels formed under varying crosslinking conditions. FIG. 16A is aschematic showing burst pressure setup for measuring the leakingpressure of the explanted rabbit cornea with an incisional perforationof 2 mm in diameter, after the bioadhesive was applied andphotocrosslinked. Tuning the visible light exposure time (i.e., 1, 2 and4 min.) generates hydrogels with varying burst pressure (FIG. 16B). Theconcentration of GelMA was 20% (w/v).

FIG. 17 is a photograph showing ex vivo assessment of smoothness,transparency and retention.

FIG. 18 is a picture showing slit lamp photographs and OCT imagesimmediately after ex vivo application of GEL-CORE to rabbit cornea and14 and 28 days later showing excellent retention.

FIGS. 19A-19F are images showing in vivo application of the bioadhesiveto corneal defects in rabbits. One day after application, thebioadhesive was transparent with a smooth surface without any cornealinflammation (FIG. 19A). There was an epithelial defect over thebioadhesive (FIG. 19B) and AS-OCT showed complete adhesion of theimplant to the cornea (FIG. 19C). One week after the application, thebioadhesive was still transparent with no associated stromal infiltratebased on slit lamp exam (FIG. 19D). Epithelial migration over thebioadhesive was evident in fluorescein staining (FIG. 19E) as well as inAS-OCT (arrows, FIG. 19F), which also showed no gap between thebioadhesive and the stroma.

FIGS. 20A-20C are representative H&E histopathology images (200×) fromrabbit corneas 2 weeks after creating a 50%-depth stromal defect (FIG.20A, normal uninjured cornea). When a corneal defect is left to healwithout bioadhesive, significant epithelial hyperplasia filling thestromal defect was observed (FIG. 20B). In contrast, when the stromaldefect is filled with GelMA bioadhesive, the biomaterial is covered byepithelial cells and is retained by the cornea, filling the entiredefect (FIG. 20C).

FIG. 21 is a graph showing ¹HNMR spectrums of GelMA prepolymer and GelMAhydrogels (20% (w/v)) formed at varying visible light exposure times,including 0, 1, 2, and 4 mm.

FIG. 22 is a bar graph showing quantification of GelMA hydrogel degreeof crosslinking, engineered by using 10 and 20% (w/v) prepolymerconcentrations at varying visible light exposure times (1, 2, and 4 min)based on ¹HNMR spectrums.

FIG. 23 is a bar graph showing ex vivo retention time of GelMA onexplanted rabbit eyes after 18 days of incubation in PBS at 4° C.

FIGS. 24A-24D show a schematic diagram showing the use of adhesive forrapid and long-term repair of corneal injuries.

DETAILED DESCRIPTION

The inventors have developed and optimized specific formulations of abioadhesive hydrogel for corneal applications: methacryloyl gelatin(GelMA) hydrogel for Corneal Reconstruction (hereafter referred to asGel-CORE). To form Gel-CORE, a natural polymer was used, gelatin, whichis derived from hydrolyzed collagen, maintaining similar bioactivity ascollagen. Gelatin was chemically functionalized with methacryloyl groupsto form a light activated and adhesive hydrogel, GelMA, with tunablephysical properties. This hydrogel can be applied to the cornea andphotopolymerized with visible light in a few seconds to form a highlyadhesive hydrogel. Specific formulations were developed with desiredbioadhesiveness, bioactivity and degradation profiles suitable forcorneal applications.

Although widespread in biomedical applications, UV light crosslinkinghas potential biosafety concerns as it may lead to undesired DNA damageand ocular toxicity. GelMA comprises modified natural extracellularmatrix components that can be crosslinkcd via visible light exposure tocreate an elastic and biodegradable hydrogel for corneal reconstructionand repair (Gel-CORE). Natural extracellular matrix components mayinclude gelatin derived from animals including, but not limited to, pig,cow, horse, chicken, fish, etc. Advantageously, the gelatin can beharvested under sterile conditions from animals in pathogen-free barrierfacilities to eliminate the risk of transmission of disease (e.g.,hepatitis C, human immunodeficiency virus, etc.)

In situ photopolymerization of GelMA facilitates easy delivery totechnically demanding locations such as the cornea, and allows forcuring of the bioadhesive exactly according to the required geometry ofthe tissue to be sealed, which is an advantage over pre-formedmaterials, as e.g., scaffolds or sheets. Besides physicalinterconnection of the curing bioadhesive with the tissue surface,gelatin offers additional options to interact with tissues in defectareas. Since gelatin contains multiple domains that bind to cell-surfacereceptors and extracellular matrix proteins, initial connection of thebioadhesive to corneal tissue as well as subsequent cell attachment toand cell growth on the bioadhesive are promoted.

As used herein, “methacryloyl gelatin” is defined as gelatin having freeamines and/or free hydroxyls that have been substituted with at leastone methacrylamide group and/or at least one methacrylate group. Gelatincomprises amino acids, some of which have side chains that terminate inamines (e.g., lysine, arginine, asparagine, glutamine) or hydroxyls(e.g., serine, threonine, aspartic acid, glutamic acid). One or more ofthese terminal amines and/or hydroxyls can be substituted withmethacryloyl groups to produce methacryloyl gelatin comprisingmethacrylamide and/or methacrylate groups, respectively. In someembodiments, with exposure to visible light in the presence of aphotoinitiator, the methacryloyl groups on one gelatin molecule canreact with the methacryloyl groups on another gelatin molecule tocrosslink the methacryloyl gelatin and produce a hydrogel. In someembodiments, the gelatin may be functionalized with methacryloyl groupsby reacting gelatin with suitable reagents including, but not limitedto, methacrylic anhydride, methacryloyl chloride, etc.

Certain exemplary embodiments of the present invention comprise aphotoinitiator. “Photoinitiator” as used herein refers to any chemicalcompound, or a mixture of compounds, that decomposes into free radicalswhen exposed to light. Preferably, the photoinitiator produces freeradicals when exposed to visible light. Exemplary ranges of visiblelight useful for exciting a visible light photoinitiator include green,blue, indigo, and violet. Preferably, the visible light has a wavelengthin the range of 450-550 nm. Examples of photoinitiators include, but arenot limited to, Eosin Y, triethanolamine, vinyl caprolactam,dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propadione(PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO),bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819),4,4′-bis(dimethylamino)benzophenone, 4,4′-bis(diethylamino)benzophenone,2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone,phenanthrenequinone, ferrocene, diphenyl(2,4,6trimethylbenzoyl)phosphine oxide/2-hydroxy-2-methylpropiophenone (50/50blend), dibenzosuberenone, (benzene) tricarbonylchromium, resazurin,resorufin, benzoyltrimethylgermane (Ivocerin®), derivatives thereof,combinations thereof, etc.

In some embodiments, the photoinitiator is a mixture of Eosin Y,triethanolamine, and vinyl caprolactam. In some embodiments, theconcentration of Eosin Y is between 0.0125 and 0.5 mM, and/or theconcentration of triethanolamine is between 0.1 and 2% (w/v), and/or theconcentration of vinyl caprolactam is between 0.05 and 1.5% (w/v). Insome embodiments, the concentration of Eosin Y is between 0.025 and 0.15mM, and/or the concentration of triethanolamine is between 0.2 and 1.6%(w/v), and/or and the concentration of vinyl caprolactam is between 0.09and 0.8% (w/v). In some embodiments, the concentration of Eosin Y isbetween 0.025 and 0.15 mM, and/or the concentration of triethanolamineis between 0.2 and 1.6% w/v, and/or the concentration of vinylcaprolactam is between 0.09 and 0.8% (w/v). In some embodiments, theconcentration of Eosin Y is between 0.05 and 0.08 mM, and/or theconcentration of triethanolamine is between 0.4 and 0.8% (w/v), and/orthe concentration of vinyl caprolactam is between 0.18 and 0.4% (w/v).In some embodiments, the concentration of Eosin Y is about 0.05 mM,and/or the concentration of triethanolamine is about 0.4% (w/v), and/orthe concentration of vinyl caprolactam is about 0.4% (w/v). In someembodiments, the concentration of Eosin Y is between 0.5 and 0.5 mM,and/or the concentration of triethanolamine is between 0.5 and 2% (w/v),and/or the concentration of vinyl caprolactam is between 0.5 and 1.5%(w/v). In some embodiments, the concentration of Eosin Y is about 0.1mM, the concentration of triethanolamine is about 0.5% (w/v), and theconcentration of vinyl caprolactam is about 0.5% (w/v).

The mechanical properties of Gel-CORE can be tuned for variousapplications by changing the degree of methacryloyl substitution, GelMAconcentration, amount of photoinitiators, and light exposure time. Asused herein, the degree of methacryloyl substitution is defined as thepercentage of free amines or hydroxyls in the gelatin that have beensubstituted with methacryloyl groups. In some embodiments,methacryloyl-substituted gelatin has a degree of methacryloylsubstitution between 20% and 90%, 30% and 85%, 50% and 90%, 60% and 85%,65% and 75%, or 70 and 80%. In some embodiments, themethacryloyl-substituted gelatin comprises methacrylamide substitutionand methacrylate substitution, and the ratio of methacrylamidesubstitution to methacrylate substitution is between 80:20 and 99:1,between 90:10 and 98:2, or between 93:7 and 97:3.

As used herein, the concentration of methacryloyl-substituted gelatin isdefined as the weight of methacryloyl-substituted gelatin divided by thevolume of solvent (w/v), expressed as a percentage. The solvent may be apharmaceutically acceptable carrier. In some embodiments, themethacryloyl-substituted gelatin is present at a concentration between5% and 25% (w/v), between 17% and 25% (w/v), between 17% and 23% (w/v),or about 20% (w/v). In some embodiments, the methacryloyl-substitutedgelatin is present at a concentration between 5% and 15% (w/v), between8% and 12% (w/v), or about 10% (w/v). In some embodiments, themethacryloyl-substituted gelatin is present at a concentration between10% and 40% (w/v), 15% and 35% (w/v), 20% and 30% (w/v), or about 5%,10%, 15%, 20%, or 25% (w/v).

In some embodiments, the methacryloyl-substituted gelatin has acombination of any of the above degrees of methacryloyl substitution andany of the above concentrations, e.g., a degree of methacryloylsubstitution between 50% and 90% and a concentration between 10% and 40%(w/v), a degree of methacryloyl substitution between 60% and 85% and aconcentration between 20% and 30% (w/v), a degree of methacryloylsubstitution between 70% and 80% and a concentration of 25% (w/v),degree of methacryloyl substitution between 30% and 85% and aconcentration between 5% and 15% (w/v), a degree of methacryloylsubstitution between 60% and 85% and a concentration between 8% and 12%(w/v), or a degree of methacryloyl substitution between 70% and 80% anda concentration of about 10% (w/v).

Certain exemplary embodiments of the present invention comprise apharmaceutically acceptable carrier. “Pharmaceutically acceptablecarrier” as used herein refers to a pharmaceutically acceptablematerial, composition, or vehicle that is involved in carrying ortransporting a compound of interest from one tissue, organ, or portionof the body to another tissue, organ, or portion of the body. Forexample, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of the formulationand is compatible with administration to a subject, for example a human.It must also be suitable for use in contact with any tissues or organswith which it may come in contact, meaning that it must not carry a riskof toxicity, irritation, allergic response, immunogenicity, or any othercomplication that excessively outweighs its therapeutic benefits.Examples of pharmaceutically acceptable carriers include, but are notlimited to, a solvent or dispersing medium containing, for example,water, pH buffered solutions (e.g., phosphate buffered saline (PBS),HEPES, TES, MOPS, etc.), isotonic saline, Ringer's solution, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol, and thelike), alginic acid, ethyl alcohol, and suitable mixtures thereof. Insome embodiments, the pharmaceutically acceptable carrier can be a pHbuffered solution (e.g. PBS) or water.

Corneal cells may be incorporated in or on the surface of thebioadhesive in order to promote corneal tissue formation and healing.Thus, in some embodiments, the GelMA or Gel-CORE composition furthercomprises corneal cells, preferably epithelial cells, endothelial cells,keratocytes, or a combination thereof. Epithelial and/or endothelialcells are preferably seeded on the surface of the composition, whilekeratocytes are preferably mixed into the composition prior tophotopolymerization.

In order to promote healing and regrowth of the cornea, to prevent ortreat infections or immune response, to prevent or treat corneal vesselformation, to treat increased intraocular pressure, or to promotegeneral eye health, the compositions of the present invention mayfurther comprise a therapeutic agent. Non-limiting examples oftherapeutic agents include an antibacterial, an anti-fungal, ananti-viral, an anti-acanthamoebal, an anti-inflammatory, animmunosuppressive, an anti-glaucoma, an anti-VEGF, a growth factor, orany combination thereof. Non-limiting examples of antibacterial agentsinclude: penicillins, cephalosporins, penems, carbapenems, monobactams,aminoglycosides, sulfonamides, macrolides, tetracyclins, lincosides,quinolones, chloramphenicol, vancomycin, metronidazole, rifampin,isoniazid, spectinomycin, trimethoprim sulfamethoxazole, chitosan,ansamycins, daptomycin, nitrofurans, oxazolidinones, bacitracin,colistin, polymixin B, and clindamycin. Non-limiting examples ofanti-fungal agents include: amphotericin B, natamycin, candicin,filipin, hamycin, nystatin, rimocidin, voriconazole, imidazoles,triazoles, thiazoles, allylamines, echinocandins, benzoic acid,ciclopirox, flucytosine, griseofulvin, haloprogin, tolnaftate,undecylenic acid, and povidone-iodine. Non-limiting examples ofanti-viral agents include: acyclovir, valacyclovir, famciclovir,penciclovir, trifluridine, and vidarabine. Non-limiting examples ofanti-acanthamoebal agents include: chlorohexidine, polyhexamethylenbiguanide, propamidine, and hexamidine. Non-limiting examples ofanti-inflammatory agents include: corticosteroids; non-steroidalanti-inflammatory drugs including salicylates, propionic acidderivatives, acetic acid derivatives, enolic acid derivatives,anthranilic acid derivatives, selective cox-2 inhibitors, andsulfonanilides; biologicals including antibodies (such as tumor necrosisfactor-alpha inhibitors) and dominant negative ligands (such asinterleukin-1 receptor antagonists). Non-limiting examples ofimmunosuppressive agents include: alkylating agents, antimetabolites,mycophenolate, cyclosporine, tacrolimus, and rapamycin. Non-limitingexamples of anti-glaucoma agents include: prostaglandin analogs, betablockers, adrenergic agonists, carbonic anhydrase inhibitors,parasympathomimetic (miotic) agents. Non-limiting examples ofanti-vascular endothelial growth factor (anti-VEGF) agents include:bevacizumab, ranibizumab, and aflibercept. Non-limiting examples ofgrowth factors include: epidermal growth factor, platelet-derived growthfactor, vitamin A, fibronectin, annexin a5, albumin, alpha-2macroglobulin, fibroblast growth factor b, insulin-like growth factor-I,nerve growth factor, and hepatocyte growth factor.

Certain aspects of the present invention are directed to a compositionfor corneal reconstruction comprising a crosslinkedmethacryloyl-substituted gelatin hydrogel and a pharmaceuticallyacceptable carrier. As used herein, a “hydrogel” is a network ofhydrophilic polymer chains forming a colloidal gel. In some embodiments,the crosslinked methacryloyl-substituted gelatin hydrogel has a degreeof methacryloyl substitution between 20% and 90%, 40% and 90%, 30 and85%, 60% and 85%, 65% and 75%, or 70% and 80%. In some embodiments, thecrosslinked methacryloyl-substituted gelatin hydrogel is present at aconcentration between 5% and 15% (w/v), 8% and 12% (w/v), 10% and 40%(w/v), 15% and 35% (w/v), 20% and 30% (w/v), or about 5%, 10%, 15%, 20%,or 25% (w/v) in the pharmaceutically acceptable carrier. In someembodiments, the crosslinked methacryloyl-substituted gelatin hydrogelhas a combination of any of the above degrees of methacryloylsubstitution and any of the above concentrations. In some embodiments,the crosslinked methacryloyl-substituted gelatin hydrogel has a degreeof methacryloyl substitution between 60% and 80% and a concentrationbetween 10% and 40% (w/v) in the pharmaceutically acceptable carrier, adegree of methacryloyl substitution between 65% and 75% and aconcentration between 20% and 30% (w/v), a degree of methacryloylsubstitution between 68% and 72% and a concentration of 25% (w/v), adegree of methacryloyl substitution between 30% and 85% and aconcentration between 5% and 15% (w/v), a degree of methacryloylsubstitution between 60% and 85% and a concentration between 8% and 12%(w/v), or a degree of methacryloyl substitution between 70% and 80% anda concentration of about 10% (w/v).

The physical properties (degradation and mechanical properties, etc.) ofGel-CORE can be modified so that different compositions of thebioadhesive can be made for different purposes, e.g., a bioadhesive witheither short or long retention time, appropriate for different clinicalscenarios. For example, in the case of a corneal trauma with extrudedintraocular contents such as iris, one may wish to apply Gel-CORE fortemporary sealing of the injured eye. In patients with cornealepithelial defects, Gel-CORE with short retention time may also be usedto cover the epithelial defect. In contrast, in the case of a corneawith a structural defect or severe thinning, Gel-CORE can be formulatedin a way that it retains for prolonged periods. Currently availablesealant technologies (e.g. cyanoacrylate) do not offer such control inthe characteristics of the final product. The following are desiredphysical properties, either alone or in combination, for bioadhesivecompositions suitable for corneal repair. In some embodiments, thecomposition has a Young's Modulus of 95-100 kPa, 110-140 kPa, or 190-260kPa. In some embodiments, the composition has an elastic modulus of 5-10kPa, 10-20 kPa, 25-80 kPa, 5-50 kPa, 5-28 kPa, 10-22 kPa, or 14-18 kPa.In some embodiments, the composition has a compressive modulus of 1-55kPa, 3-160 kPa, 5-320 kPa, 10-250 kPa, 25-200 kPa, 50-175 kPa or 75-150kPa. In some embodiments, the composition has a wound closure strengthof ≥40 kPa, ≥50 kPa, ≥60 kPa, ≥70 kPa, ≥80 kPa, ≥90 kPa or ≥100 kPameasured using the Wound Closure test (ASTM F2458-05). In someembodiments, the composition has a shear resistance strength of ≥100kPa, ≥150 kPa, or ≥200 kPa, measured using the Lap Shear test (ASTMF2255-05). In some embodiments, the composition has a burst pressure of≥15 kPa, ≥17 kPa, or ≥20 kPa, measured using the Burst Pressure test(ASTM F2392-04).

Certain aspects of the present invention are directed to methods forcorneal reconstruction, comprising the steps of:

-   -   a) applying a composition comprising a methacryloyl-substituted        gelatin, a visible light activated photoinitiator, and a        pharmaceutically acceptable carrier to a corneal defect; and    -   b) exposing the composition to visible light.

The mechanical properties of Gel-CORE can be tuned for variousapplications by changing the visible light exposure time. Without beingbound by theory, longer visible light exposure time produces morecrosslinkage in the methacryloyl-substituted gelatin, providing ahydrogel with improved mechanical properties, such as adhesion strength,shear strength, compressive strength, tensile strength, etc. In someembodiments, the composition is exposed to visible light for a timeperiod between 30 seconds and 6 minutes, between 1 minute and 5 minutes,between 2 minutes and 4 minutes, or 3 minutes. In some embodiments, thecomposition is exposed to visible light for a time period of less thanone minute, within 10-60 seconds, 15-45 seconds, 20 seconds, or 30seconds. In some embodiments, the composition is exposed to visiblelight for a time period between 20 and 120 seconds, or between 30 and 60seconds. In some embodiments, the composition can be exposed to visiblelight for a time period between 60 seconds and 240 seconds. In someembodiments, the composition can be exposed to visible light for a timeperiod of about 60 seconds, about 120 seconds, about 180 seconds orabout 240 seconds.

In some embodiments, the method does not comprise suturing the cornea.Exemplary ranges of visible light useful for crosslinking the Gel-MAcomposition include green, blue, indigo, and violet. Preferably, thevisible light has a wavelength in the range of 450-550 nm.

Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%,±4%, ±4.5%, or ±5%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

As used herein the terms “comprising” or “comprises” means “including”or “includes” and are used in reference to compositions, methods,systems, and respective component(s) thereof, that are useful to theinvention, yet open to the inclusion of unspecified elements, whetheruseful or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, systems, andrespective components thereof as described herein, which are exclusiveof any element not recited in that description of the embodiment.

The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

Exemplary embodiments of the invention can be described by one or moreof the following sub paragraphs:

-   -   1. A composition for corneal reconstruction comprising a        methacryloyl-substituted gelatin (gelatin methacryloyl), a        visible light activated photoinitiator, and a pharmaceutically        acceptable carrier.    -   2. The composition of paragraph 1, wherein the        methacryloyl-substituted gelatin has a degree of methacryloyl        substitution between 30% and 85%.    -   3. The composition of paragraph 1 or 2, wherein the        methacryloyl-substituted gelatin has a degree of methacryloyl        substitution between 60% and 85%.    -   4. The composition of any one of paragraphs 1-3, wherein the        methacryloyl-substituted gelatin has a degree of methacryloyl        substitution between 70% and 80%.    -   5. The composition of any one of paragraphs 1-4, wherein the        methacryloyl-substituted gelatin comprises methacrylamide        substitution and methacrylate substitution, and the ratio of        methacrylamide substitution to methacrylate substitution is        between 80:20 and 99:1.    -   6. The composition of paragraph 5, wherein the ratio of        methacrylamide substitution to methacrylate substitution is        between 90:10 and 98:2.    -   7. The composition of paragraph 5 or 6, wherein the ratio of        methacrylamide substitution to methacrylate substitution is        between 92:8 and 97:3.    -   8. The composition of any one of paragraphs 1-7, wherein the        gelatin methacryloyl is present at a concentration between 5%        and 25% (w/v).    -   9. The composition of any of paragraphs 1-8, wherein the gelatin        methacryloyl is present at a concentration between 17% and 25%        (w/v).    -   10. The composition of any of paragraphs 1-9, wherein the        gelatin methacryloyl is present at a concentration between 17%        and 23% (w/v).    -   11. The composition of any of paragraphs 1-10, wherein the        gelatin methacryloyl is present at a concentration of about 20%.    -   12. The composition of any one of paragraphs 1-8, wherein the        gelatin methacryloyl is present at a concentration between 8%        and 12% (w/v).    -   13. The composition of paragraph 12, wherein the gelatin        methacryloyl is present at a concentration of about 10% (w/v).    -   14. The composition of any one of paragraphs 1-13, wherein the        visible light activated photoinitiator is selected from the        group consisting of: Eosin Y, triethanolamine, vinyl        caprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ),        1-phenyl-1,2-propadione (PPD),        2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO),        bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide        (Ir819), 4,4′-bis(dimethylamino)benzophenone,        4,4′-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one,        4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene,        diphenyl(2,4,6 trimethylbenzoyl)phosphine        oxide/2-hydroxy-2-methylpropiophenone (50/50 blend),        dibenzosuberenone, (benzene) tricarbonylchromium, resazurin,        resorufin, benzoyltrimethylgermane, derivatives thereof, and any        combination thereof.    -   15. The composition of any of paragraphs 1-14, wherein the        visible light activated photoinitiator comprises a mixture of        Eosin Y, triethanolamine, and vinyl caprolactam.    -   16. The composition of paragraph 15, wherein the concentration        of Eosin Y is between 0.0125 and 0.5 mM, and/or the        concentration of triethanolamine is between 0.1 and 2% (w/v),        and/or the concentration of vinyl caprolactam is between 0.05        and 1.5% (w/v).    -   17. The composition of paragraph 15 or 16, wherein the        concentration of Eosin Y is between 0.025 and 0.15 mM, and/or        the concentration of triethanolamine is between 0.2 and 1.6%        (w/v), and/or and the concentration of vinyl caprolactam is        between 0.09 and 0.8% (w/v).    -   18. The composition of any of paragraphs 15-17, wherein the        concentration of Eosin Y is between 0.025 and 0.15 mM, and/or        the concentration of triethanolamine is between 0.2 and 1.6%        (w/v), and/or the concentration of vinyl caprolactam is between        0.09 and 0.8% (w/v).    -   19. The composition of any of paragraphs 15-18, wherein the        concentration of Eosin Y is between 0.05 and 0.08 mM, and/or the        concentration of triethanolamine is between 0.4 and 0.8% (w/v),        and/or the concentration of vinyl caprolactam is between 0.18        and 0.4% (w/v).    -   20. The composition of any of paragraphs 15-19, wherein the        concentration of Eosin Y is about 0.05 mM, and/or the        concentration of triethanolamine is about 0.4% (w/v), and/or the        concentration of vinyl caprolactam is about 0.4% (w/v).    -   21. The composition of paragraph 15 or 16, wherein the        concentration of Eosin Y is between 0.5 and 0.5 mM, and/or the        concentration of triethanolamine is between 0.5 and 2% (w/v),        and/or the concentration of vinyl caprolactam is between 0.5 and        1.5% (w/v).    -   22. The composition of any of paragraphs 15, 16 or 21, wherein        the concentration of    -   Eosin Y is about 0.1 mM, the concentration of triethanolamine is        about 0.5% (w/v), and the concentration of vinyl caprolactam is        about 0.5% (w/v).    -   23. The composition of any one of paragraphs 1-12, further        comprising corneal cells.    -   24. The composition of paragraph 23, wherein the corneal cells        comprise epithelial cells, endothelial cells, keratocytes, or a        combination thereof.    -   25. The composition of any one of paragraphs 1-24, wherein the        composition further comprises a therapeutic agent.    -   26. The composition of paragraph 25, wherein the therapeutic        agent is selected from the group consisting of an antibacterial,        an anti-fungal, an anti-viral, an anti-acanthamoebal, an        anti-inflammatory, an immunosuppressive, an anti-glaucoma, an        anti-VEGF, a growth factor, and any combination thereof.    -   27. A composition for corneal reconstruction comprising a        crosslinked gelatin methacryloyl hydrogel and a pharmaceutically        acceptable carrier, wherein the crosslinked        methacryloyl-substituted gelatin (gelatin methacryloyl) hydrogel        has a degree of methacryloyl substitution between 30% and 85%        and a concentration between 5% and 25% (w/v) in the        pharmaceutically acceptable carrier.    -   28. The composition of paragraph 27, wherein the concentration        is between 5% and 15% (w/v).    -   29. The composition of paragraph 27 or 28, wherein the        crosslinked gelatin methacryloyl hydrogel has a degree of        methacryloyl substitution between 60% and 85% and a        concentration between 8% and 12% (w/v).    -   30. The composition of any of paragraphs 27-29, wherein the        crosslinked gelatin methacryloyl hydrogel has a degree of        methacryloyl substitution between 70% and 8% and a concentration        of about 10% (w/v).    -   31. The composition of paragraph 27, wherein the concentration        is between 17% and 25% (w/v).    -   32. The composition of paragraph 27 or 28, wherein the        crosslinked gelatin methacryloyl hydrogel has a degree of        methacryloyl substitution between 60% and 85% and/or a        concentration between 17% and 23% (w/v).    -   33. The composition of any of paragraphs 27-29, wherein the        crosslinked gelatin methacryloyl hydrogel has a degree of        methacryloyl substitution between 70% and 80% and/or a        concentration of about 20% (w/v).    -   34. The composition of any one of paragraphs 27-33, having a        Young's Modulus of 190-260 kPa or 250-350 kPa.    -   35. The composition of any one of paragraphs 27-33, having a        Young's Modulus of 110-140 kPa or 100-150 kPa.    -   36. The composition of any one of paragraphs 27-35, having an        elastic modulus of 5-50 kPa.    -   37. The composition of any one of paragraphs 27-36, having a        compressive modulus of 5-320 kPa or 10-250 kPa.    -   38. The composition of any one of paragraphs 27-37, having a        compressive modulus of 5-160 kPa or 125-175 kPa.    -   39. The composition of any one of paragraphs 27-38, having a        wound closure strength of ≥40 kPa.    -   40. The composition of any one of paragraphs 27-38, having a        wound closure strength of ≥45 kPa.    -   41. The composition of any one of paragraphs 27-40, having a        burst pressure of ≥10 kPa or ≥15 kPa.    -   42. The composition of any one of paragraphs 27-41, wherein the        composition further comprises a therapeutic agent.    -   43. The composition of paragraph 42, wherein the therapeutic        agent is selected from the group consisting of an antibacterial,        an anti-fungal, an anti-viral, an anti-acanthamoebal, an        anti-inflammatory, an immunosuppressive, an anti-glaucoma, an        anti-VEGF, a growth factor, and any combination thereof.    -   44. The composition of any one of paragraphs 27-43, further        comprising corneal cells.    -   45. The composition of paragraph 44, wherein the corneal cells        comprise epithelial cells, endothelial cells, keratocytes, or a        combination thereof.    -   46. The composition of any one of paragraphs 27-45, which is        substantially transparent.    -   47. The composition of any one of paragraphs 27-46, comprising a        substantially smooth surface.    -   48. A method for corneal reconstruction, comprising the steps        of:        -   a. applying a composition comprising a            methacryloyl-substituted gelatin, a visible light activated            photoinitiator, and a pharmaceutically acceptable carrier to            a corneal defect; and        -   b. exposing the composition to visible light.    -   49. The method of paragraph 48, wherein the        methacryloyl-substituted gelatin has a degree of methacryloyl        substitution between 30% and 85%.    -   50. The method of paragraph 48 or 49, wherein the        methacryloyl-substituted gelatin has a degree of methacryloyl        substitution between 60% and 85%.    -   51. The method of any one of paragraphs 48-50, wherein the        methacryloyl-substituted gelatin has a degree of methacryloyl        substitution between 70% and 80%.    -   52. The method of any one of paragraphs 48-51, wherein the        methacryloyl-substituted gelatin comprises methacrylamide        substitution and methacrylate substitution, and the ratio of        methacrylamide substitution to methacrylate substitution is        between 80:20 and 99:1.    -   53. The method of paragraph 52, wherein the ratio of        methacrylamide substitution to methacrylate substitution is        between 90:10 and 98:2.    -   54. The method of paragraph 52 or 53, wherein the ratio of        methacrylamide substitution to methacrylate substitution is        between 92:8 and 97:3.    -   55. The method of any one of paragraphs 48-54, wherein the        methacryloyl-substituted gelatin is present at a concentration        between 5% and 25% (w/v).    -   56. The method of any one of paragraphs 48-55, wherein the        methacryloyl-substituted gelatin is present at a concentration        between 17% and 25% (w/v).    -   57. The method of any one of paragraphs 48-56, wherein the        methacryloyl-substituted gelatin is present at a concentration        between 17% and 23% (w/v).    -   58. The method of any one of paragraphs 48-57, wherein the        methacryloyl-substituted gelatin is present at a concentration        of about 20%.    -   59. The method of any one of paragraphs 48-55, wherein the        methacryloyl-substituted gelatin is present at a concentration        between 8% and 12% (w/v).    -   60. The method of paragraph 59, wherein the        methacryloyl-substituted gelatin is present at a concentration        of about 10% (w/v).    -   61. The method of any one of paragraphs 48-60, wherein the        visible light activated photoinitiator is selected from the        group consisting of: Eosin Y, triethanolamine, vinyl        caprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ),        1-phenyl-1,2-propadione (PPD),        2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO),        bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide        (Ir819), 4,4′-bis(dimethylamino)benzophenone,        4,4′-bis(diethylamino)benzophenone, 2-chlorothioxanthen-9-one,        4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene,        diphenyl(2,4,6 trimethylbenzoyl)phosphine        oxide/2-hydroxy-2-methylpropiophenone (50/50 blend),        dibenzosuberenone, (benzene) tricarbonylchromium, resazurin,        resorufin, benzoyltrimethylgermane, derivatives thereof, and any        combination thereof.    -   62. The method of any one of paragraphs 48-61, wherein the        visible light activated photoinitiator comprises a mixture of        Eosin Y, triethanolamine, and vinyl caprolactam.    -   63. The method of paragraph 62, wherein the concentration of        Eosin Y is between 0.0125 and 0.5 mM, and/or the concentration        of triethanolamine is between 0.1 and 2% w/v, and/or the        concentration of vinyl caprolactam is between 0.05 and 1.5% w/v.    -   64. The method of paragraph 62 or 63, wherein the concentration        of Eosin Y is between 0.025 and 0.15 mM, and/or the        concentration of triethanolamine is between 0.2 and 1.6% w/v,        and/or and the concentration of vinyl caprolactam is between        0.09 and 0.8% w/v.    -   65. The method of any of paragraphs 62-64, wherein the        concentration of Eosin Y is between 0.025 and 0.15 mM, and/or        the concentration of triethanolamine is between 0.2 and 1.6%        w/v, and/or the concentration of vinyl caprolactam is between        0.09 and 0.8% w/v.    -   66. The method of any of paragraphs 62-65, wherein the        concentration of Eosin Y is between 0.05 and 0.08 mM, and/or the        concentration of triethanolamine is between 0.4 and 0.8% w/v,        and/or the concentration of vinyl caprolactam is between 0.18        and 0.4% w/v.    -   67. The method of any of paragraphs 62-66, wherein the        concentration of Eosin Y is about 0.05 mM, and/or the        concentration of triethanolamine is about 0.4% w/v, and/or the        concentration of vinyl caprolactam is about 0.4% w/v.    -   68. The method of paragraph 62 or 63, wherein the concentration        of Eosin Y is between 0.5 and 0.5 mM, and/or the concentration        of triethanolamine is between 0.5 and 2% w/v, and/or the        concentration of vinyl caprolactam is between 0.5 and 1.5% w/v.    -   69. The composition of any of paragraphs 62, 63 or 68, wherein        the concentration of Eosin Y is about 0.1 mM, the concentration        of triethanolamine is about 0.5% w/v, and the concentration of        vinyl caprolactam is about 0.5% w/v.    -   70. The method of any one of paragraphs 48-69, wherein the        composition is exposed to visible light with a wavelength in the        range of 450 to 550 nm.    -   71. The method of any one of paragraphs 48-70, wherein the        composition is exposed to visible light for a time period        between 20 and 120 seconds.    -   72. The method of any one of paragraphs 48-71, wherein the        composition is exposed to visible light for a time period        between 30 and 60 seconds.    -   73. The method of any one of paragraphs 48-72, wherein the        composition further comprises a therapeutic agent.    -   74. The method of paragraph 73, wherein the therapeutic agent is        selected from the group consisting of an antibacterial, an        anti-fungal, an anti-viral, an anti-acanthamoebal, an        anti-inflammatory, an immunosuppressive, an anti-glaucoma, an        anti-VEGF, a growth factor, and any combination thereof.    -   75. The method of any one of paragraphs 48-74, wherein the        composition further comprises corneal cells.    -   76. The method of paragraph 75, wherein the corneal cells        comprise epithelial cells, endothelial cells, keratocytes, or a        combination thereof.    -   77. The method of any one of paragraphs 48-76, wherein the        composition is substantially transparent.    -   78. The method of any one of paragraphs 48-77, wherein the        method does not comprise suturing the cornea.

It is noted that the invention provides an improved bioadhesive forrepair and reconstruction of defects and injuries to the cornea.Following ASTM standard tests, crosslinked methacryloyl-substitutedgelatin hydrogels of the present invention (Gel-CORE) were shown toexhibit adhesive properties, i.e. wound closure strength, shearresistance and burst pressure, that were suitable for application to thecornea. In vitro experiments showed that Gel-CORE is cytocompatible withcorneal cells and promotes cell integration after application. In vivoexperiments in rabbits showed that Gel-CORE can effectively seal cornealdefects. Advantageously, the bioadhesives of the present invention arelow cost, easy to produce, and easy to use, making them a promisingsubstance to be used for corneal repair, as well as an easily tunableplatform to further optimize the adhesive characteristics.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

EXAMPLES

The disclosure is further illustrated by the following examples whichshould not be construed as limiting. The examples are illustrative only,and are not intended to limit, in any manner, any of the aspectsdescribed herein. The following examples do not in any way limit theinvention.

Example 1: Advantages and Applications of Gel-CORE

Gel-CORE bioadhesive may advantageously be used for the followingapplications:

Filling Corneal Defects:

Gel-CORE can fill corneal defects and corneal thinning disorders, whichoccur not only from physical injuries but also after a wide array ofcorneal inflammatory disorders, such as microbial keratitis andimmune-mediated corneal melts. In such cases, the bioadhesive is used toprovide structural support in the emergency setting. In addition, inlong-term it allows regeneration of corneal cells into the bioadhesive,which acts as a stromal replacement technology without the need to usethe traditional treatment modalities such as corneal transplantation andtissue patch graft.

The current standards of care for repair of corneal stromal defects andthinning include tissue/patch grafting or glue application. Cornealtransplantation and patch grafting require donor tissues, which may notbe available. In addition, the use of allogeneic tissues for graftingcarries a risk for immune reactions.

Currently, there is no approved adhesive for filling corneal defects.Although cyanoacrylate glue is currently used as “off-label” fortreating many emergent ophthalmic settings such as corneal perforations,impending perforations and progressive corneal thinning disorders, ithas several major drawbacks, including:

-   -   i. Low biocompatibility, with cytotoxic effects on the cornea        and other ocular tissues (risk of cataract formation and        retinopathy if it enters the eye);    -   ii. Lack of transparency, precluding good vision and impairing        view of retrocorneal structures;    -   iii. Risk of secondary infection due to high porosity;    -   iv. Difficult to control its application, with glue potentially        falling off unpredictably;    -   v. Rough surface requiring contact lens wear, which adds        additional infection risk; and    -   vi. Does not integrate with corneal tissue.

In contrast, Gel-CORE has several advantages as compared to currentlyavailable adhesives for corneal repair and sealing.

1. Biosafety:

Gel-CORE has superior biocompatibility since its base material isgelatin, which is a naturally derived biopolymer from collagen that hasbeen used in different medical applications, raising little safetyconcerns over other materials (e.g. cyanoacrylate-based products). Inaddition, by selecting a visible light activated photoinitiator, thepotential damage associated with UV exposure in previous formulationswill be avoided.

2. Tunable Properties:

the physical properties (degradation and mechanical properties, etc.) ofGel-CORE can be modified so that different compositions of thebioadhesive can be made for different purposes—a bioadhesive with eithershort or long retention time, appropriate for different clinicalscenarios. For example, in the case of a corneal trauma with extrudedintraocular contents such as iris, one may wish to apply Gel-CORE fortemporary sealing of the injured eye. In contrast, in the case of acornea with a structural defect or severe thinning, Gel-CORE can beformulated in a way that it retains for prolonged periods. Currentlyavailable sealant technologies (e.g. cyanoacrylate) do not offer suchcontrol in the characteristics of the final product.

3. Transparency:

in ex vivo experiments with the rabbit cornea, Gel-CORE has beendemonstrated to be transparent with a smooth and convex surface that canretain the normal corneal contour after administration andphotopolymerization on a 50%-deep corneal injury (FIG. 1A-1E).

4. Reversibility:

unlike cyanoacrylate where the application leads to immediate hardening,Gel-CORE requires application of light for hardening; thus amisapplication can be reversed if needed.

5. High Adhesion and Retention:

Gel-CORE has high adhesion to tissues based on wound closure, lap shear,burst pressure, and ex vivo adhesion tests. In addition, ex vivo datahas consistently shown that Gel-CORE is retained for many days with thebioadhesive remaining uncompromised and completely attached to thecornea.

6. Corneal Tissue Regeneration:

unlike the other adhesives used for cornea sealing (e.g. cyanoacrylate),Gel-CORE permits both tissue sealing and regeneration. Human cornealkeratocytes can grow within Gel-CORE. Gel-CORE will adhere to the corneastrongly, and be retained while the tissue is undergoing physiologicalrepair/regeneration.

Sealing Corneal, Limbal, or Scleral Wounds:

The traditional treatment for such wounds includes suturing. However,suturing is associated with substantial drawbacks which include thefollowing: 1. Sutures are foreign bodies that can serve as risk factorsfor microbial entrapment and infection, inflammation, andneovascularization. 2. Corneal sutures often induce regular or irregularastigmatism, leading to impaired visual acuity. To avoid thesedrawbacks, sealants have been used to seal wounds. The only approvedsealants ReSure® (in the U.S.) and OcuSeal® (in the Europe) are forsealing corneal incisions of cataract surgery. However, they fall offquickly. In contrast, Gel-CORE which strongly seals the corneal, limbal,or scleral wounds can be tuned to provide the sealing for a desiredlonger time. Gel-CORE can provide adhesion for closure of cornealwounds. In such conditions, it allows sealing the wound without the needfor suturing. Gel-CORE can provide adhesion for closure of limbal andscleral wounds. In such conditions, it allows sealing the wound withoutthe need for suturing.

Covering Corneal Epithelial Defects:

The traditional treatment for patients with corneal epithelial defectincludes eye patching, bandage contact lens, and sometime an invasiveprocedure. However, these options are limited by the fact that they canbe bothersome for the patient and can increase the risk of cornealinfections. For these cases, in contrast, we can use a fast-degradingformulation of Gel-CORE to protect the cornea while corneal epitheliumregenerates itself.

Temporary Protection of Intraocular Structures in Cases with Corneal orScleral Lacerations and Prolapse of Intraocular Structures:

The only available treatment option for such cases is surgicalrepositioning of intraocular structures with suturing the wounds whichshould be performed by a skilled surgeon in an equipped facility.However, this will delay the surgical procedure which predisposes thepatient to intraocular infection. In contrast, the use of Gel-COREprovides a temporary support for cornea/sclera and intraocularstructures while preventing infection. A permanent repair can later beperformed without imposing a high risk of intraocular infection.Gel-CORE can be used in cases with extensive corneal/scleral injuriesassociated with iris/choroid-retina prolapse as a therapeutic agent(e.g. antibiotic, etc.)-containing biologic patch to cover intraocularstructures. In such cases, it protects the intraocular structures andprevents infection before a permanent surgical procedure is performed.

Corneal Infections with or without Significant Thinning:

The current standard of care for corneal infection includes frequentinstillation of eye drops, which is cumbersome for the patient. To avoidthis drawback, currently there are some studies using contact lenseswith slow-release antibiotic. However, such technologies do not provideany structural support for the cornea. In contrast, Gel-CORE not onlycan provide extended release of antibiotic(s) but also provide thestructural support for the cornea with infectious keratitis. Gel-COREcan be used in cases of corneal infections with and without significantthinning as an antibiotic-containing patch which provides an extendedrelease of antibiotic(s) in addition to a structural support for thecornea.

Inflammatory Corneal Thinning:

The current standard of care for inflammatory corneal thinning includesuse of topical or systemic anti-inflammatory medications. Forsignificant thinning, a surgical procedure is performed as describedabove for corneal stromal defects. In contrast, Gel-CORE not onlyprovides structural support as described above for corneal defects, butalso can act as a drug reservoir to slowly release anti-inflammatorymedications, thereby obviating or reducing the need for additionaltopical or systemic medications. Thus, Gel-CORE can be used as aplatform for drug delivery as the bioadhesive is clear and can beretained for many weeks based on the therapeutic use. Gel-CORE can beused in cases of inflammatory corneal thinning as ananti-inflammatory-containing patch, which provides an extended releaseof anti-inflammatory medication(s) in addition to providing structuralsupport for the cornea.

Refractive Corneal Modeling:

Although various intracorneal implants have previously been used forrefractive modeling of the cornea, including PermaVision (ReVisionOptics), Kamra (AcoFocus), Flexivue Microlens (Presbia), and Raindropinlay (Revision Optics), they are all associated with deposit or hazeformation due to lack of complete biocompatibility with the cornealtissue. In contrast, Ge-CORE has high degree of biocompatibility whichprevents it from these complications. In addition, in Gel-CORE there isintegration of the corneal cells with the biomaterial which will nothappen in other inlays. Gel-CORE can be used as an intracorneal implantfor corneal modeling to change the refractive power of the cornea inpatients with refractive error (myopia, hyperopia, astigmatism, andpresbyopia).

Replacement of Corneal Tissue in Transplantation:

Although various artificial corneas have previously been used to replacethe corneal tissue in transplantation, including BostonKeratoprosthesis, osteoodentokeratoprosthesis, AlphaCor, they sufferfrom the fact that there is no integration of corneal cells into theseartificial corneas. In contrast, Gel-CORE shows a high degree ofmigration and integration of native corneal cells into the biomaterial.Gel-CORE can be used as a replacement of corneal tissue in lamellarcorneal transplantation instead of using donor corneal tissue. Gel-COREcan also be used as a replacement of corneal tissue in full-thicknesscorneal transplantation (similar to artificial cornea).

Example 2: Synthesis of Gelatin Methacryloyl (GelMA) Prepolymer

GelMA was synthesized as previously described.¹⁴ Concentrations ofgelatin and methacrylic anhydride may be varied to produce GelMA havingthe ranges of methacryloyl substitution disclosed herein. For example,10% (w/v) porcine gelatin (Sigma-Aldrich, St. Louis, Mo., USA) wasdissolved in phosphate-buffered saline (PBS) and heated at 60° C. for 20minutes. Dropwise addition of 8% (v/v) methacrylic anhydride(Sigma-Aldrich, St. Louis, Mo., USA) under continuous stirring at 50° C.for 3 hours was followed by dilution with PBS and dialysis againstdeionized water at 40-50° C. for 7 days. After sterile filtration andlyophilization for 4 days, GelMA was stored at −80° C. untilexperimental use.

Quantification of Methacrylamide Groups.

Conversion of amine groups in biomaterials such as GelMA has beenconventionally determined using a proton nuclear magnetic resonance (¹HNMR) spectrum.¹³⁻¹⁴ However, since gelatin is a mixture of polypeptideswith complicated compositions, it might not be feasible to detect anddifferentiate the resonance peaks from methacrylamide and methacrylategroups from ¹H NMR spectra. Instead, a fluoraldehyde assay⁴³ allows foreasier and more accurate determination of the conversion of aminegroups. When the modified protein/peptide samples are mixed with theassay solution containing o-phthalaldehyde and 2-mercaptoethanol, allthe remaining primary amine groups in the materials will be convertedinto fluorescent species with blue emissions (FIG. 2A). Four distinctGelMA formulations were prepared that vary in their degree ofmethacryloyl substitution by adding different amounts of MA to thereaction (Table 1).¹⁴ Depending on the degree of modification, theresulting GelMA formulations will be referred to as ultra-GelMA,high-GelMA, medium-GelMA, and low-GelMA, respectively. Using gelatin asthe standard, the amount of remaining primary amine groups can be easilyobtained. The conversions of amine groups of the resulting GelMA sampleswere determined using the fluoraldehyde assay as ˜93% for ultra-GelMA,˜84% for high-GelMA, ˜65% for medium-GelMA, and ˜24% for low-GelMA,respectively (FIG. 2B). It is clear that the conversion of amine groupsis positively correlated with the added MA amount at a fixed reactiontemperature and reaction time.

TABLE 1 Summary of molecular parameters of different GelMA samples.Conversion Estimated amount Estimated amount of of amine ofmethacrylamide methacrylate groups Preparation Samples groups groups(mmol/g) (mmol/g) conditions Type-A 93% 0.46^(a) 0.034 20% (v/v) MA,Ultra-GelMA 50° C., 3 h Type-A 84% 0.42^(a) 0.028 8% (v/v) MA,High-GelMA 50° C., 3 h Type-A 65% 0.32^(a) 0.022 5% (v/v) MA,Medium-GelMA 50° C., 3 h Type-A 24% 0.12^(a) 0.008 0.5% (v/v) MA,Low-GelMA 50° C., 3 h

Quantification of Methacrylate Groups.

In our previous publications on preparation of GelMA,¹⁴ ¹HNMR spectrawere used to determine the conversion of amine groups by calculationsbased on the integration areas of the resonance peak from the aminegroups. Quantification of the methacrylate groups was unable to performdue to the lack of distinguishable resonance peaks of the hydroxylgroups in ¹HNMR spectra of the modified peptide or protein samples.

Here, a Fe(III)-hydroxamic acid-based assay was used to determine theamount of methacrylate groups in different GelMA samples (FIG. 3A-3E).Hydroxamic acid forms a brown-red complex with Fe(III) ions, which canserve as a qualitative test for hydroxamic acid species (FIG. 3A). Thisclass of complexes has an absorption peak centered at around 500 nm(FIGS. 3B and 3C). Formation of the Fe(III)-hydroxamic acid complexeshas been used to quantify the reactivity of amine groups and hydroxylgroups of lysozymes with several different carboxylic acid anhydrides,⁴⁴along with other analytic applications such as the quantification ofester group residues in poly(vinyl alcohol).⁴⁵ Acetohydroxamic acid(AHA) was used as the standard to establish the working curve and it isassumed that the complex of acetohydroxamic acid and Fe(III) ions(FeAHA) have similar spectrophotometric properties with that ofN-hydroxymethacrylamide and Fe(III) ions (FeHMA).⁴⁴ Iron(III)perchlorate was dissolved in dilute hydrochloric acid to prepare theFe(III) ion solutions, which were added to the acetohydroxamic acidsolutions in large excess to form a 1:1 complex. It has been reportedthat the apparent extinction coefficient reaches its maximum when themolar ratio of Fe(III) and hydroxamic acid was over 20 and will remainindependent on the ratio⁴⁶. UV-Vis absorption spectra of the series ofstandard FeAHA solutions were recorded in UV-transparent microplatecovering the concentrations of from 1.3×10⁻⁴ to 2.5×10⁻³ mol/L. Indeed,excellent linearity was achieved when the absorbance was plotted as afunction of AHA concentration and analyzed with a linear least-squarefit (FIG. 3D).

To determine the amount of methacrylate groups in GelMA samples, anaminolysis reaction to convert the methacrylate groups to the detectableN-hydroxymethacrylamide compound was employed. In particular, GelMAsamples at 50 mg/mL were treated with hydroxylamine solutions at roomtemperature for 10 min to generate N-hydroxymethacrylamide. Theresulting solution was acidified with hydrochloric acid, followed by theaddition of excess Fe(III) ions. Color change upon the addition ofFe(III) ions indicated the formation of the FeHMA complex, whichconfirmed the existence of methacrylate groups. Concentrations of theFeHMA complex formed in situ were determined from the UV-Vis absorptionspectra, which could be used to calculate the amounts of methacrylategroups in the GelMA samples (FIG. 3E). For all tested GelMA samples, itwas found that methacrylate groups represented below 10% of allmethacryloyl substitutions. These results suggested that the aminegroups are indeed more reactive than the hydroxyl groups, and themethacrylamide groups are the dominant form in GelMA (FIG. 3E).

Example 3: Preparation and Material Characterization of Gel-COREHydrogels

Visible light crosslinkable Gel-CORE was made by using Eosin-Y(Sigma-Aldrich) as a visible-light activated initiator, triethanolamine(TEOA) (Sigma-Aldrich) as a co-initiator, and vinyl caprolactam (VC)(Sigma-Aldrich) as a catalyst.^(15,16) Using this crosslinking system,polymerization of methacryloyl groups on GelMA was initiated throughexposure to blue light (450-550 nm, Xenon source) at 100 mW/cm² (FIGS.4A-4C). Eosin-Y is a water-soluble xanthene dye and is a common stainfor collagen, the main component of the cornea and sclera. This visiblelight system has a well-established track record of biocompatibility ina range of applications¹⁵⁻¹⁹ and has gained FDA approval for use in thehuman body in the polyethylene glycol (PEG)-based sealant FocalSeal®(Genzyme Biosurgical, Cambridge, Mass.). Visible light crosslinkableGel-CORE hydrogels show tunable physical properties (FIGS. 5A-5B); theelastic modulus of Gel-CORE hydrogels using visible light crosslinkingcould be tuned from 5-28 kPa (FIGS. 5A-5B). In addition, the swellingratio could be changed from 7% to 13% (w/w) (FIG. 6B).

To form the hydrogels, GelMA with different degrees of methacryloylmodification (30%-85%) can be used.¹⁴ Then, different concentrations ofGelMA prepolymer solutions (5-15% w/v) can be prepared in phosphatebuffered saline (PBS) containing Eosin-Y (0.1-0.5 mM), TEOA (0.5-2%w/v), and VC (0.5-1.5% w/v). The formulated GelMA prepolymer solutionscan be photocrosslinked by exposure to blue light for 20-120 seconds,which matches the absorption spectrum of Eosin-Y (FIG. 4B). Varying theGelMA concentration, Eosin-Y/TEOA/VC concentrations, and light exposuretime varies the physical properties of the engineered hydrogels.

As the formulations are used to repair the cornea, the Gel-COREhydrogels should have similar elasticity and stiffness to the nativecornea (Young Modulus: 250-350 kPa). The swelling ratio of the hydrogelshould be optimized to obtain swelling ratio of <20% to ensure that theadhesive preserves its shape after being applying in the corneal defect.In particular, the swelling ratio can affect the shape, curvature andthe smoothness of the sealed defect.

For example, freeze-dried GelMA produced according to Example 2 wasdissolved in PBS at a concentration of 10% (w/v). After addition of aphotoinitiator mixture of 0.1% (w/v) Eosin Y, 0.5% (w/v)triethanolamine, and 0.5% (w/v) vinyl caprolacatam and dissolving at 80°C., the prepolymer solution was photocrosslinked to a hydrogel(Gel-CORE) by visible light irradiation (450-550 nm, Xenon source, 100mW/cm²).

In another example, different concentrations of GelMA (5, 10, 15, 20%(w/v)) were tested for material characterization. Freeze-dried GelMA (asproduced in Example 2) was dissolved in PBS containing 1.875% (w/v)triethanolamine (TEA) and 1.25% (w/v) N-vinylcaprolactam (VC) atconcentrations of 5, 10, 15, 20% (w/v). Eosin Y was separately dissolvedin fresh DPBS at a concentration of 0.5 mM. To prepare the hydrogel, 8μL, of GelMA solution was mixed with 2 μL of Eosin Y solution, and thenthe mixture was placed between two glass coverslips separated by 150 μmspacers, followed by exposure to blue-green light (100 mW/cm², Xenonsource from Genzyme Biosurgery) in the range of 450 to 550 nm for 20sec.

Mechanical testing of Gel-CORE samples was conducted as previouslypublished.¹⁴ Briefly, prepolymer solution was photocrosslinked toproduce the following geometries: discs for compressive testing (n=3 to5; 6 mm in diameter and 1.5 mm in height) and cuboids for tensiletesting (n=7 to 10; 3 mm in width, 14 mm in length and 1.5 mm inthickness). The hydrogels were either directly analyzed or stored in PBSat 4° C. for 24 hours before being examined on an mechanical testingsystem 5542 (Instron, Norwood, Mass., USA). The strain rate was set to 1mm/min for compressive testing and tensile testing. The compressivestrength and the ultimate tensile strength of the samples weredetermined at the point of breaking or tearing of the hydrogels. Thecompressive modulus and elastic modulus were obtained by measuring theslope of stress/strain curves at strain rate between 0-0.5%.

In order to analyze the swelling characteristics, Gel-CORE hydrogelsamples (n=5) were allowed to swell in PBS for 1, 2 or 3 days. At theend of the experiment, excess liquid was gently removed with a tissue,and the wet weight was measured. After lyophilization, the dry weight ofthe samples was measured, and the swelling ratio was calculated as (wetweight-dry weight)/dry weight (FIGS. 6A and 6B).

Ex vivo test for Gel-CORE hydrogels used explanted rabbit corneatissues. GelMA prepolymer was first applied on incision created on theexplanted cornea and then photocrosslinked by exposure to visible lightusing optimized light exposure time. The burst pressure was thenmeasured by using a pressure sensor after air inflation into the cornea.For example, a rabbit cornea was sealed with Gel-CORE (10% (w/v),prepolymer concentration, 5 mM Eosin-Y, and exposure time of 120 sec.The incision created on rabbit cornea was tightly and completely sealedwith Gel-CORE and the tissue could be pressurized up to around 3.5 kPa(26 mmHg), which is double of the pressure of healthy eye. Preferably,Gel-CORE samples have a burst pressure higher than 15 kPa (>110 mmHg),²⁰⁻²¹ a lap shear strength >100 kPa, adhesion strength >40 kPa, andphotopolymerize in <60 seconds of light exposure.

Example 4: ASTM Standard In Vitro Testing of the Mechanical Propertiesof Gel-CORE Burst Pressure

The burst pressure testing of sealants was adapted from the ASTMstandard F2392-04 (standard test method for burst strength of surgicalsealants). Porcine skin sheets (40 mm*40 mm) were soaked in PBS prior tosample preparation. A circular defect (3 mm in diameter) was created inthe center of a pig skin sheet that was placed between two Teflon sheets(35 mm*35 mm). The top Teflon sheet contained a hole (10 mm in diameter)to allow for application of the desired adhesive over the circulardefect in the porcine skin sheet (FIG. 7A). In the case of GelMA, theprepolymer was irradiated with visible light. Afterwards, the collagensheet was removed and placed into the burst pressure testing system,consisting of pressure detection and recording unit and a syringe pump,which applied air with continuously increasing pressure towards thesamples (FIG. 7B). Each tested adhesive group contained five samples.

Increasing air pressure was applied on sealant covering a standardizeddefect in porcine skin to test the burst pressure resistance. Each GelMAconcentration resulted in higher burst pressure values than Coseal™(FIG. 7C).

Wound Closure

The wound closure strengths of GelMA and the clinically establishedsurgical sealants Evicel® (Ethicon, Somerville, N.J., USA), Coseal™(Baxter, Deerfield, Ill., USA) and Progel were examined referring to theASTM standard test F2458-05 (standard test method for wound closurestrength of tissue adhesives and sealants), whereas the standard methodwas slightly modified to fit a smaller sample size. In brief, freshporcine skin from a local slaughterhouse was prepared by removing theadipose tissue layer and cutting the sample into rectangular sectionsmeasuring 5 mm*15 mm. While unused, porcine skin was kept moist in gauzesoaked in PBS. Before use, porcine skin was blotted dry to remove excessliquid, and each end of the skin strip was fixed onto two poly(methylmethacrylate) slides (30 mm*60 mm) with Krazy glue (Westerville, Ohio,USA), leaving a 6 mm section of skin between the slides. The porcineskin strip was then cut apart using a razor blade (FIG. 8A), andpetroleum jelly was applied with a syringe to the ends of the desiredadhesive application area. Afterwards, 40 μl of the adhesive was appliedacross the 6 mm*5 mm skin section and, in the case of GelMA, irradiatedwith visible light (FIG. 8A). After 1 hour of incubation in PBS, the twoplastic slides were placed into the Instron system grips for tensiletesting (FIG. 8A). The adhesive strength of a sealant sample wasdetermined at the point of tearing. Each tested adhesive group containedfour to seven samples and results are summarized in FIG. 8B. The tensiletest to measured elastic modulus (ranged from 5-50 kPa) and ultimatetensile strength (stress at break point after stretching sample).

Example 5: Degradation and Retention of Gel-CORE

Gel-CORE was applied to a 3-mm >50%-deep corneal defect (10% (w/v)Gel-CORE solution containing 0.01% (w/v) Eosin-Y, 0.5% (w/v) TEA, and0.5% (w/v) VC was used). The solution was exposed to blue light for 120seconds to form a hydrogel layer on the damaged cornea. After theprocedure, eyes were kept in PBS at 4° C. Changes in Gel-CORE over timewere assessed using serial evaluations with slit lamp biomicroscopy andOCT. It was noted that for at least 11 days, the bioadhesive remaineduncompromised (full thickness and spread retained) and stayed completelyattached to the cornea in all tested eyes. Slit lamp biomicroscopyshowed that during this time, the bioadhesive remained clear with asmooth surface without any biomicroscopic signs of changes in shape orcontour (FIGS. 9A and 9C). In addition, OCT confirmed no change in thethickness or shape of Gel-CORE (FIGS. 9B and 9D). After 11 days, thecorneal tissue in PBS started to degrade (as expected from necrosis dueto prolonged storage in PBS), at which point the Gel-CORE attachment tothe cornea began to get compromised.

A corneal injury model in New Zealand white rabbits was used by creatinga 50%-deep corneal defect. After general anesthesia of the rabbit usingintramuscular injection of ketamine and xylazine, a circular 50%-deepcorneal defect was created in the right eye by a 3-mm biopsy punch.Then, a surgical crescent knife was used to perform a lamellarkeratectomy. After removing the anterior lamella, the defect surface wasdried using a surgical microsponge. Then, 10 μl of the bioadhesivesolution was instilled to fill the corneal defect. A microsponge wasthen used to smooth over the extra solution. This was immediatelyfollowed by blue light application (using FocalSeal Xenon Light Source,Genzyme, 100 w/cm²) for 120 seconds to crosslink the bioadhesive. Thedegradation and retention of Gel-CORE was evaluated using slit lampbiomicroscopy and OCT at 1, 2, and 4 weeks, as described below.

Two outcome measures were evaluated over 4 weeks of follow-up: (i)bioadhesive transparency which measures optical degradation (asevaluated by slit lamp biomicroscopy using Fantes grading scale,²² whichis based on visibility of iris details); and (ii) bioadhesive thickness(as measured by OCT, described below).

Retention is a function of two parameters, degradation and adhesiveness.Either degradation and/or suboptimal adhesiveness can lead to loss ofgel retention. To measure retention, OCT technology was used, asdescribed below, to evaluate (i) the presence of the bioadhesivecovering the corneal defect; and (ii) the thickness of any gap betweenthe bioadhesive and corneal epithelium or stroma over 4 weeks offollow-up.

Slit lamp biomicroscopy and OCT imaging were performed under generalanesthesia for both eyes of the rabbit at 1-week follow-up andsubsequently only for the operated eye. For slit lamp examinations, aTopcon Slit Lamp system was used. With a 25× magnification and usingslit and broad beams, transparency of the bioadhesive was evaluated(using the Fantes grading scale). Slit lamp photographs were alsoobtained at the time of examination. Optical Coherence Tomography (OCT)was also employed: this is a non-contact imaging modality that provideshigh-resolution cross-sectional images of the cornea in vivo. In thisexperiment, a spectral-domain OCT (Spectralis, Heidelberg Engineering,GmbH, Germany), which has an axial resolution of 3.9-7 μm, was used.Line scans (8 mm long) were performed at 0, 45, 90, and 135 degrees inthe central cornea (FIG. 10A). Using the proprietary software of theOCT, the thickness (in microns) of the bioadhesive (in the operated eye)and of the cornea (in the unoperated fellow eye) was measured in thecenter of the cornea and at 1 mm away from the center in both directions(FIG. 10B). In addition, the thickness of any gap between thebioadhesive and corneal tissue was measured in microns. The slit lampand OCT findings were compared between the two eyes, and between thedifferent time points to determine the degradation of Gel-CORE and itsretention in the corneal defect over time. Based on preliminary data,Gel-CORE can remain intact in vitro for at least 30 days.

Example 6: Biocompatibility and Integration of Gel-CORE

The optimal bioadhesive for cornea repair is not only non-toxic forcorneal cells, but also permits cells to integrate into the biomaterialfor long-term integration and to prevent extrusion. The in vitrocytocompatibility and integrative capacity of Gel-CORE was determined byusing the two most abundant cell types in the cornea includingkeratocytes and corneal epithelial cells. Keratocytes and cornealepithelial cells were cultured using 2D and 3D culture systems. Thebiocompatibility and integrative capacity of Gel-CORE was assessed invivo by investigating the effects of the bioadhesive on corneal cells,as well as the migration of corneal cells into the bioadhesive overtime.

In preliminary experiments, the compatibility of Gel-CORE with cornealcells was demonstrated (FIGS. 11A-11G). Corneal keratocytes wereincorporated within a representative Gel-CORE composition, showing ≥95%cell survival using 2D culture systems, as well as proliferation andmigration of the keratocytes when grown either on top or within theGel-CORE construct (FIG. 11G).

In Vitro Evaluation of Gel-CORE Cytocompatibility and Cell Integration.

To evaluate the in vitro cytocompatibility of Gel-CORE for the cornea,the following experiments were performed. Corneal cells were cultured ina 5% CO₂ humidified incubator at 37° C. in culture media (Dulbecco'sModified Eagle's Medium (DMEM) containing 10% Fetal bovine serum, 1%penicillin-streptomycin, and 1% glutamic acid). A 2D culture system wasused in which epithelial cells were seeded on the top of Gel-CORE toform epithelial monolayers. Moreover, a 3D culture system was used toencapsulate the keratocytes inside the Gel-CORE to form cornea tissue.

For 2D culture, Gel-CORE was constructed following exposure to visiblelight as detailed herein. Then, the gels were seeded with the epithelialcells at cell densities ranging from 1×10⁶ to 1×10⁸ cells/mL and wereincubated for 14 days. Media was changed every other day. Cell viabilitywas evaluated on days 1, 4, 7 and 14 by using calcein-AM/ethidiumhomodimer Live/Dead assays.²³ Actin/DAPI staining was used to assesscellular attachment and spreading as explained previously.^(13,24-26) Inaddition, the metabolic activities of the cells were assessed by usingPrestoBlue assay followed by absorbance readings on a microplatespectrophotometer on days 1, 4, 7 and 14. In addition, cellularinfiltration and growth within the hydrogels were investigated byhistology analyses on day 14.²⁷⁻²⁹ Moreover, K12 expression was analyzedfor the corneal epithelial cells due to its acclaimed role inmaintaining corneal epithelial function. It is critical that epithelialcells grow on the surface of the hydrogel (without penetration into thegel) to form a dense cell layer, which is required for eye protection.Based on these in vitro experiments, Gel-CORE is shown to benon-cytotoxic (cell viability >90%) and promote cellular metabolicactivity and adhesion and have limited penetration in the gel.

For 3D culture, keratocytes were mixed with GelMA prepolymer solution atconcentrations ranging from 1×10⁶-1×10⁸ cells/mL. The mixture was thenexposed to light to form cell-laden Gel-CORE adhesive. The gels was thenwashed 3 times with PBS and incubated for 14 days in medium in a cultureincubator at 37° C. Cellular viability (Live/Dead assay), cellattachment and spreading (Actin/DAPI), proliferation (Picogreen assay),collagen deposition (Picrosirius Red), and corneal tissue formation(Haematoxylin and eosin staining) was assessed on days 1, 4, 7, and 14.Based on these 3D studies, Gel-CORE was shown to be cytocompatible andwill promote cornea tissue formation.

In Vivo Biocompatibility and Integrative Capacity of Gel-CORE in theCornea.

A Corneal injury model in New Zealand white rabbits was used by creatinga 50%-deep corneal defect, as described herein. Rabbits were dividedinto three groups: (i) Gel-CORE group, in which the bioadhesive was usedto fill the corneal defect, as described herein; (ii) Cyanoacrylategroup, in which cyanoacrylate glue, which is the standard of care(albeit unapproved) for filling corneal defects to prevent perforation,was used. For this, 10 μl cyanoacrylate glue (MSI-EpiDermGlu+Flex,Medisav Services, Canada) was applied to fill the corneal defect,followed immediately by placement of a soft-bandage contact lens overthe cornea. (iii) Control group, in which corneal defect was not filledby any adhesive but received prophylactic antibiotic (erythromycin)ointment for 1 week. The rabbits were then followed for 12 weeks. Thebiocompatibility and integrative capacity of Gel-CORE and the degree ofcorneal inflammation and neovascularization were evaluated and comparedto other groups using slit lamp biomicroscopy and IVCM (as describedbelow) at 1, 2, 4, and 12 weeks. In addition, at each time point 6rabbits per group were sacrificed to harvest the cornea for histologic(n=3) and immunohistochemical evaluations (n=3).

For biocompatibility evaluation, the following were considered as theoutcome measures: (i) transparency of the cornea surrounding theadhesive/defect (evaluated by slit lamp biomicroscopy using Fantesgrading scale); and (ii) density of epithelial cells, stromalkeratocytes, inflammatory cells, and blood vessels in the cornea aroundthe bioadhesive (measured by IVCM, histologic staining, and/orimmunohistochemical staining, as detailed below).

For evaluation of integrative capacity, the following were considered asthe outcome measures: (i) transparency of the bioadhesive (evaluated byslit lamp biomicroscopy using Fantes grading scale); (ii) the extent ofmigration of corneal epithelial cells over the bioadhesive (measured byslit lamp biomicroscopy, IVCM, and histologic staining, as detailedbelow), and (iii) density of stromal keratocytes, corneal nerves,inflammatory cells, and blood vessels within the bioadhesive/cornealdefect area (measured by IVCM, histologic staining, and/orimmunohistochemical staining, described below).

Slit lamp biomicroscopy and IVCM were performed under generalanesthesia. As described herein, slit lamp examination and photographywere used to assess transparency of the cornea and the bioadhesive. Inaddition, to assess the migration of epithelium over the bioadhesive,slit lamp photography with fluorescein staining was performed, and thearea of corneal epithelial defect over the bioadhesive was measuredusing ImageJ's Measure Area tool for each time point. In Vivo ConfocalMicroscopy (IVCM) was employed to evaluate cellular changes andmigration in the same rabbits over time without sacrificing the animal.This is a non-invasive imaging modality which provides high-resolutionimages at the cellular level from the cornea in live animals (FIGS.12A-12F). In this experiment, a laser scanning IVCM (Heidelberg RetinaTomograph 3 with Rostock Cornea Module, Heidelberg, Germany) was usedwhich utilizes a 670 nm diode laser and has a resolution of 1 μm. Itprovides images that represent a corneal area of 400×400 μm. ForIVCM-based readouts, the following were scaned and examined: (i) the 1.5mm-central cornea (over the bioadhesive, cyanoacrylate glue, or originaldefect in the control group); and (ii) the corneal tissue surroundingthe 1 mm circumference of the adhesive in 4 quadrants (superior, nasal,inferior, and temporal). For scanning, Sequence Mode was used whichautomatically acquires 100 consecutive images per sequence. With manualadvancing, all corneal layers (epithelium, stroma, and endothelium) wereimaged in each scan. Two Sequence Mode scans were obtained in each offive locations (central, superior, nasal, inferior, and temporal). Forimage analysis in the central cornea, 5 images were randomly selectedfrom different depths in each scan (totally 10 images). For imageanalysis in the cornea around the adhesive, one randomly selected imagefrom each corneal layer (epithelium, subbasal layer, and stroma) wasselected from each individual sequence scan (totally 8 images perlayer). For analysis, the density of epithelial cells, subbasalinflammatory cells, and stromal keratocytes were measured by a maskedobserver using Image J software as previously reported.³⁰⁻³⁵ Inaddition, the density of corneal nerves was also assessed by a maskedobserver using NeuronJ software as previously reported.^(30, 36, 37)

Histologic evaluation using hematoxylin and eosin (H&E) staining wasperformed on cryosections of the harvested corneas. From each cornea, 5sections were obtained from the central cornea containing both thedefect/adhesive location and the surrounding corneal tissue. For imageanalysis, all sections were evaluated by a masked observer. Migration ofcorneal epithelial cells over the adhesive was determined. In addition,the density of stromal keratocytes and inflammatory cells was determinedin 10 randomly selected areas within the adhesive (both in the centerand periphery of the adhesive) in addition to 10 randomly selected areasof the surrounding cornea at 200 μm from the margin of the adhesive.

Immunohistochemical staining was also performed on cryosections of theharvested cornea with antibodies against the following: β-tubulin III(2G10 Ab; Abcam), beta 2 (CD18) integrin (L13/64 for inflammatory cells;GeneTex), and CD31 (polyclonal anti-CD31 for blood vessels; Abcam). Thedensity of these cells was determined by a masked observer in both theadhesive-applied and surrounding corneal matrix as described for the H&Estaining. For this, serial sections from 10 randomly selected areaswithin the adhesive as well as 10 randomly selected areas of thesurrounding cornea within 200 μm of the adhesive were used for analysis.Mean and Standard Deviation (SD) was measured for each metric.

The slit lamp, IVCM, histologic, and immunohistochemical findings werecompared between the three groups at each time point to determine thebiocompatibility and integrative capacity of Gel-CORE for fillingcorneal defects. These comparisons between the Gel-CORE group and thecontrol group helped determine whether Gel-CORE caused more or lessinflammation and tissue damage than expected from secondary-intentionhealing (which is also included as a control). In addition, comparisonsbetween the Gel-CORE group and the cyanoacrylate group showed whetherthe potential tissue damage is less in the Gel-CORE group compared tothe current standard of care adhesive. In each group, comparisonsbetween different time points showed whether the integration of cornealcells into Gel-CORE developed over time and whether any potential tissuedamage caused by the adhesive subsided or aggravated over time.

Example 7: A New Bioadhesive for Rapid and Long-Term Repair of CornealStromal Defects

Use of Tunable Properties of Bioadhesive to Optimize its Physical andAdhesive Properties.

The data shows that the GelMA prepolymer with 80-90% degree ofmethacryloyl functionalization can be effectively crosslinked by usingEosin Y as a photosensitizer, triethanolamine (TEOA) as an initiator,and vinyl caprolactam (VC) as a catalyst to form a stable hydrogel withtunable physical properties. As the crosslinking efficacy is dependenton the concentration of the photosensitizer, initiator, and catalyst,systematic optimization of these conditions is essential. By tuning theconcentration of Eosin Y, TEOA, and VC, the critical mechanicalproperties of the hydrogel can be precisely controlled to deriveformulations with tensile and compressive moduli that are comparable tothe native cornea (FIGS. 14A-14C, 15A and 15B). Based on the data,optimized concentrations of Eosin Y (0.05 mM), TEOA (0.4% w/v), and VC(0.4% w/v), are used for the following experiments.

Adhesive Properties of Engineered Bioadhesives.

A standard burst pressure test was used to obtain a comprehensiveestimation of the sealing ability of 20% w/v visible light crosslinkedGelMA hydrogels formed at various visible light exposure times. The exvivo tests were performed to measure the burst pressures of rabbitcorneas with 2-mm full-thickness incisions (FIGS. 16A and 16B). Theburst pressure of the engineered GelMA was higher than 200 mmHg, almost10 times the pressure of a healthy eye, and significantly higher thanthe burst pressure of the commercial control, ReSure® (FIG. 16B).

Ex Vivo Assessment of Smoothness, Transparency, and Retention.

Ex vivo tests were performed using explanted rabbit corneas to assessthe GelMA bioadhesives. The bioadhesive was applied ex vivo to a3-mm >50%-deep corneal defect in New Zealand rabbit eyes. For this, a20% w/v GelMA solution containing 0.05 mM Eosin-Y, 0.4% w/v TEOA, and0.4% w/v VC was used. The solution was exposed to visible light for 120sec to form a hydrogel layer on the corneal defect showing firm adhesionof the bioadhesive to the corneal stroma. In addition, the bioadhesivewas transparent with a smooth surface as shown in FIG. 17.

After the procedure, the eyes were kept in PBS at 4° C. Changes in thebioadhesive over time were assessed using serial evaluations with slitlamp biomicroscopy and Anterior Segment Optical Coherence Tomography(AS-OCT). It was noted that for the duration of a 30-day assessmentperiod the bioadhesive remained uncompromised (thickness and spread werefully retained) and stayed completely attached to the cornea in alltested eyes. Slit lamp biomicroscopy also showed that during this timethe bioadhesive remained clear with a smooth surface without anybiomicroscopic signs of changes in shape or contour (FIG. 18). Inaddition, AS-OCT confirmed no change in the thickness or shape of thebioadhesive.

In Vivo Assessment of Biocompatibility and Biointegration.

A corneal injury model in New Zealand white rabbits was used by creatinga 50%-deep corneal defect. After general anesthesia using intramuscularinjection of ketamine and xylazine, a central 50%-deep corneal cut wascreated in the right eye followed by application of the bioadhesive.Immediately after photocrosslinking, there was a firm adhesion of thebioadhesive to the corneal defect. One day after surgery (FIG. 19A-19C),the bioadhesive was transparent with a smooth surface, and thesurrounding cornea was transparent and non-inflamed. AS-OCT also showedcomplete adhesion to the stromal bed. One week after surgery (FIG.19D-19F), the bioadhesive was still transparent, with some migration ofthe corneal epithelium over the bioadhesive.

Histologic evaluation of harvested rabbit corneas 2 weeks afterundergoing surgery showed migration of epithelial cells over, andmigration of keratocytes into, the bioadhesive (FIG. 20C). Additionally,preliminary IHC studies showed a 20.9% decrease in CD45⁺ cellinfiltration in GelMA bioadhesive-filled corneas compared with injuredcorneas left to heal without bioadhesive.

Example 8: Crosslinking of GelMA Hydrogels

¹Hnmr Analysis.

¹H NMR analysis was performed to obtain the crosslinking degree ofgelatin methacryloyl (GelMA) hydrogels produced by using various visiblelight exposure times including 1, 2, and 4 min (FIG. 21). To perform ¹HNMR test, uncrosslinked GelMA prepolymer and GelMA hydrogels produced atvarious visible light exposure times were dissolved in deuterated DMSO.In order to quantify the degree of crosslinking, all spectrums werenormalized with respect to the phenylalanine signal (8=6.9-7.3 ppm). Itis frequently reported that the signals related to protons ofmethacrylate groups in GelMA appear at peaks located at 8=5.30 and 5.64ppm.^(47, 48) The degree of crosslinking was calculated as below:

${{Degree}\mspace{14mu}{of}\mspace{14mu}{Crosslinking}\mspace{14mu}( {D\; C} )\%} = {( {1 - \frac{{Area}( {{methacrylate}\mspace{14mu}{groups}} )}{{Area}( {{phenylalanine}\mspace{14mu}{signal}} )}} ) \times 100}$which represents the ratio of remaining C═C in the methacrylated groupsafter the crosslinking process.

¹Hnmr Results.

Based on ¹HNMR analysis, the degree of crosslinking was calculated fromdisappearance of the C═C bond correlated to methacrylated group at8=5.30 and 5.64 ppm. The degree of crosslinking for 20% (w/v) GelMAhydrogels increased from 63.4±2.7 at 1 min to 88.9±7.8 at 4 mincrosslinking time, respectively (FIG. 22 and Table 2). Furthermore, for10% (w/v) GelMA concentration, after 1 min reaction time, 86.8±1.3 ofthe original methacrylated groups were consumed (FIG. 22). The degree ofcrosslinking was 90.7±0.2 and 92.9±2.2% after 2 min and 4 min,respectively.

TABLE 2 Quantification of GelMA hydrogel degree of crosslinking,engineered by using 10% and 20% (w/v) prepolymer concentrations atvarying visible light exposure times (1, 2, and 4 min) based on ¹HNMRspectrums. GelMA concentration Light exposure time (min) % (w/v) 1 2 410 86.8 ± 1.3 90.7 ± 0.2 92.9 ± 2.2 20 63.4 ± 2.7 76.2 ± 2.4 88.9 ± 7.8

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All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

What is claimed is:
 1. A composition for corneal or scleralreconstruction or repair comprising a methacryloyl-substituted gelatin,a visible light activated photoinitiator, and a pharmaceuticallyacceptable carrier, wherein the methacryloyl-substituted gelatincomprises methacrylamide substitution and methacrylate substitution, anda ratio of methacrylamide substitution to methacrylate substitution inthe methacryloyl-substituted gelatin is between 80:20 and 99:1.
 2. Thecomposition of claim 1, wherein the methacryloyl-substituted gelatin hasa degree of methacryloyl substitution between 30% and 85%.
 3. Thecomposition of claim 1, wherein the methacryloyl-substituted gelatin ispresent at a concentration between 5% and 25% (w/v).
 4. The compositionof claim 3, wherein the methacryloyl-substituted gelatin is present at aconcentration between 8% and 12% (w/v) or between 17% and 23% (w/v). 5.The composition of claim 4, wherein the methacryloyl-substituted gelatinis present at a concentration of about 10% (w/v) or of about 20% (w/v).6. The composition of claim 1, wherein the visible light activatedphotoinitiator is selected from the group consisting of: Eosin Y,triethanolamine, vinyl caprolactam,dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propadione(PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO),bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819),4,4′-bis(dimethylamino)benzophenone, 4,4′-bis(diethylamino)benzophenone,2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone,phenanthrenequinone, ferrocene, diphenyl(2,4,6trimethylbenzoyl)phosphine oxide/2-hydroxy-2-methylpropiophenone (50/50blend), dibenzosuberenone, (benzene) tricarbonylchromium, resazurin,resorufin, benzoyltrimethylgermane, and any combination thereof.
 7. Thecomposition of claim 1, wherein the visible light activatedphotoinitiator is a mixture comprising Eosin Y, triethanolamine, andvinyl caprolactam.
 8. The composition of claim 7, wherein aconcentration of Eosin Y is between 0.0125 and 0.5 mM, a concentrationof triethanolamine is between 0.1 and 2% w/v, and a concentration ofvinyl caprolactam is between 0.05 and 1.5% w/v.
 9. The composition ofclaim 8, wherein: (i) the concentration of Eosin Y is about 0.1 mM, theconcentration of triethanolamine is about 0.5% w/v, and theconcentration of vinyl caprolactam is about 0.5% w/v; or (ii) theconcentration of Eosin Y is about 0.05 mM, the concentration oftriethanolamine is about 0.4% w/v, and the concentration of vinylcaprolactam is about 0.4% w/v.
 10. The composition of claim 1, whereinthe composition further comprises corneal cells.
 11. The composition ofclaim 1, wherein the composition further comprises a therapeutic agent.12. A method for corneal or scleral reconstruction or repair, comprisingthe steps of: a. applying a composition of claim 1 to a corneal orscleral defect; and b. exposing the composition to visible light.